WO2002096926A1 - Modulateurs d'agents pharmacologiques - Google Patents

Modulateurs d'agents pharmacologiques Download PDF

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WO2002096926A1
WO2002096926A1 PCT/US2002/016555 US0216555W WO02096926A1 WO 2002096926 A1 WO2002096926 A1 WO 2002096926A1 US 0216555 W US0216555 W US 0216555W WO 02096926 A1 WO02096926 A1 WO 02096926A1
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nucleic acid
acid ligand
modulator
oligonucleotide
aptamer
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PCT/US2002/016555
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English (en)
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Bruce A. Sullenger
Christopher Rusconi
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Duke University
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Priority to DK02739409.7T priority Critical patent/DK1401853T3/da
Priority to AT02739409T priority patent/ATE482227T1/de
Priority to CA2448567A priority patent/CA2448567C/fr
Priority to JP2003500105A priority patent/JP4773684B2/ja
Priority to IL15905302A priority patent/IL159053A0/xx
Priority to DE60237768T priority patent/DE60237768D1/de
Priority to EP02739409A priority patent/EP1401853B1/fr
Priority to AU2002312059A priority patent/AU2002312059B2/en
Publication of WO2002096926A1 publication Critical patent/WO2002096926A1/fr
Priority to IL159053A priority patent/IL159053A/en
Priority to AU2009200036A priority patent/AU2009200036B2/en

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P25/00—Drugs for disorders of the nervous system
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21022—Coagulation factor IXa (3.4.21.22)
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol

Definitions

  • the present invention relates, in general, to an agent that modulates the pharmacological activity of a nucleic acid ligand (e.g., aptamer) and, in particular, to an agent that enhances or inhibits the activity of such a ligand.
  • a nucleic acid ligand e.g., aptamer
  • the invention further relates to a composition comprising such an agent and to a method of using these agents and compositions in medical therapeutic and diagnostic procedures.
  • SELEX Systematic Evolution of Ligands by Exponential enrichment
  • Nucleic acids have conventionally been thought of as primarily playing an informational role in biological processes. Through a method known as Systematic Evolution of Ligands by Exponential enrichment, termed SELEX, it has become clear that nucleic acids have three dimensional structural diversity not unlike proteins. SELEX is a method for the in vitro synthesis and selection of nucleic acid molecules with highly specific binding to target molecules. The SELEX process was first described by Gold and Tuerk in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, now U.S. Pat. No. 5,475,096, and thereafter in U.S. patent application Ser. No. 07/931,473, filed Aug.
  • Nucleic acid ligands or aptamers are nonencoding single-stranded nucleic acid (DNA or RNA) that have the property of binding specifically to a desired target compound or molecule, and that have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets.
  • the SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity.
  • the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids that have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
  • Nucleic acid ligands possess a number of features that can render them useful as therapeutic agents. They can be made as relatively small (e.g., 8 kDa to 15 kDa) synthetic compounds and can be selected to possess high affinity and specificity for target molecules (equilibrium dissociation constants ranging from, for example, 0.05-10 nM). Aptamers embody both the affinity properties of monoclonal antibodies and single chain antibodies (scFv's) and the manufacturing ease similar to that of a small peptide.
  • scFv's single chain antibodies
  • aptamers As synthetic compounds, site specific modifications (insertions or deletions) can be made to aptamers to rationally alter their bioavailability and mode of clearance. For example, it has been found that 2'-fluoro pyrimidine-modified aptamers in the 10 kDa to 12 kDa size range have a short circulating half-life ( ⁇ 10 minutes) following bolus intravenous administration but that simple chemical modification of the aptamer or conjugation of the aptamer to a high molecular weight inert carrier molecule (e.g., PEG) increases circulating half-life substantially (6-12 hours) (Willis et al, Bioconjug Chem 9:573-582 (1998), Tucker et al, J Chromatogr Biomed Sci Appl 732:203-212 (1999), Watson et al, Antisense Nucleic Acid Drug Dev 10:63-75 (2000)).
  • a high molecular weight inert carrier molecule e.g., PEG
  • L-nucleotides Bioactive and nuclease resistant single-stranded nucleic acid ligands comprising L-nucleotides have been described (Williams et al, Proc. Natl. Acad. Sci. 94:11285 (1997); USP 5,780,221; Leva et al, Chem. Biol. 9:351 (2002)). These "L-aptamers” are reportedly stable under conditions in which aptamers comprising nucleotides of natural handedness (D-nucleotides) (that is, "D-aptamers”) are subject to degradation.
  • D-nucleotides that is, “D-aptamers”
  • U.S. Patent No. 6,114,120 relates to an aptamer that binds to a cell macromolecule.
  • U.S. Patent No. 5,670,637 relates to aptamers that bind to proteins.
  • U.S. Patent No. 5,696,249 relates to an aptamer produced by the SELEX process.
  • U.S. Patent No. 5,756,291 and 5,582,981 to O'Toole disclose a method for detecting thrombin using a labeled aptamer that comprises a defined six nucleotide sequence.
  • U.S. Patent Nos. 5,527,894 and 5,637,461 of Gold et al. relate to methods of identifying aptamers against the tat protein.
  • Other patents that disclose aptamers directed against specific biological targets include U.S. Patent Nos.
  • aptamers A number of patents have also issued that relate to specific uses of aptamers.
  • Bruice et al. in U.S. Patent No. 6,022,691 describe the use of aptamers identified by a SELEX-like process to detect drugs and other molecules in biological fluids.
  • Gold etal. in U.S. Patent No. 5,843,653 provide a diagnostic method using aptamers.
  • U.S. Patent No. 6,110,900 discloses a diagnostic composition that contains an aptamer.
  • U.S. Patent No. 5,789,163 discloses a sandwich assay that employs aptamers as the capture and/or detection ligand.
  • U.S. Patent Nos. 5,670,633 and 6,005,087 to Cook et al. describe thermally stable 2'- fiuoro oligonucleotides that are complementary to an RNA or DNA base sequence.
  • U.S. Patent Nos. 6,222,025 and 5,760,202 to Cook et al. describe the synthesis of 2'-O substituted pyrimidines and oligomers containing the modified pyrimidines.
  • EP 0 593 901 Bl discloses oligonucleotide and ribozyme analogues with terminal 3 ',3'- and 5',5'-nucleoside bonds.
  • U.S. Patent No. 6,011,020 to Gold et al. discloses an aptamer modified by polyethylene glycol.
  • aptamers designed to bind to other aptamers include: Aldaz-Carroll L, Tallet B, Dausse E, Yurchenko L, Toulme JL; Apical loop-internal loop interactions: a new RNA-RNA recognition motif identified through in vitro selection against RNA hairpins of the hepatitis C virus mRNA; Biochemistry.
  • TAR trans-activation responsive
  • the present invention provides compounds, compositions and methods to improve the process of using nucleic acid ligands in medical therapy.
  • the approach provided by the present invention allows for more control over the therapeutic effect, pharmac ⁇ kinetics and duration of activity of nucleic acid ligands.
  • nucleic acid ligands can be modulated (i.e., enhanced or inhibited) in vivo to produce a desired biological effect.
  • This can be accomplished through the administration of a modulator, or regulator, that changes the binding of the nucleic acid ligand for its target or that degrades or otherwise cleaves, metabolizes or breaks down the nucleic acid ligand while the ligand is still exerting its effect.
  • Modulators of the present invention can be administered in real time as needed based on various factors, including the progress of the patient, as well as the physician's discretion in how to achieve optimal therapy.
  • this invention provides for the first time a regulatable therapeutic regime in the course of nucleic acid ligand therapy.
  • This regulatable therapeutic regime controls drug action by introducing a modulator that is easy to use, can be independent of the patient's health status, has a uniform mode of action, and does not require continuous drug infusion.
  • an antidote is provided that is rationally designed to turn off aptamer activity when desired by the physician.
  • the modulator can be a oligonucleotide, a small molecule, a peptide, oligosaccharide, for example an aminoglycoside, or other molecule that can bind to or otherwise modulate the activity of the therapeutic nucleic acid ligand, including a small molecule, or a chimera or fusion or linked product of any of these.
  • the modulator can be an oligonucleotide that is complementary to at least a portion of the nucleic acid ligand.
  • the modulator can be a ribozyme or DNAzyme that targets the nucleic acid ligand.
  • the modulator can be a peptide nucleic acid (PNA), morpholino nucleic acid (MNA), locked nucleic acid (LNA) or pseudocyclic oligonucleobases (PCO) that includes a sequence that is complementary to or hybridizes with at least a portion of the nucleic acid ligand.
  • PNA peptide nucleic acid
  • MNA morpholino nucleic acid
  • LNA locked nucleic acid
  • PCO pseudocyclic oligonucleobases
  • the mechanism of formation of a duplex between a complementary oligonucleotide modulator of the invention and a nucleic acid ligand is the same as between two short linear oligoribonucleotides, both the rules for designing such interactions and the kinetics of formation of such a product are impacted by the intramolecular aptamer structure.
  • the rate of nucleation is important for formation of the final stable duplex, and the rate of this step is greatly enhanced by targeting the oligonucleotide modulator to single-stranded loops and/or single- stranded 3* or 5' tails present in the nucleic acid ligand.
  • the free energy of formation of the intermolecular duplex has to be favorable with respect to formation of the existing intramolecular duplexes within the targeted nucleic acid ligand.
  • the modulator itself is an aptamer.
  • a nucleic acid ligand is first generated that binds to the desired therapeutic target.
  • a second nucleic acid ligand that binds to the first nucleic acid ligand is generated using the SELEX process described herein or other process, and modulates the interaction between the therapeutic nucleic acid ligand and the target.
  • the second nucleic acid ligand deactivates the effect of the first nucleic acid ligand.
  • the aptamer which binds to the target can be a PNA, MNA, LNA or PCO and the modulator is a nucleic acid ligand.
  • the aptamer which binds to the target is a PNA, MNA, LNA or PCO, and the modulator is a PNA.
  • the aptamer which binds to the target is a PNA, MNA, LNA or PCO, and the modulator is an MNA.
  • the aptamer which binds to the target is a PNA, MNA, LNA or PCO, and the modulator is an LNA.
  • the aptamer which binds to the target is a PNA, MNA, LNA or PCO
  • the modulator is a PCO. Any of these can be used, as desired, in the naturally occurring stereochemistry or in non-naturally occurring stereochemistry or a mixture thereof.
  • the nucleic acid ligand is in the D configuration, and in an alternative embodiment, the nucleic acid ligand is in the L configuration.
  • the present invention also provides methods to identify the modulators of nucleic acid ligands.
  • Modulators can be identified in general, through binding assays, molecular modeling, or in vivo or in vitro assays that measure the modification of biological function.
  • the binding of a modulator to a nucleic acid is determined by a gel shift assay.
  • the binding of a modulator to a nucleic acid ligand is determined by a Biacore assay.
  • Other appropriate assays are described in the Detailed Description of the Invention.
  • the binding or interaction of the modulator with the nucleic acid ligand is measured by evaluating the effect of the nucleic acid ligand with and without the modulator under appropriate biological conditions.
  • modulators of the invention can be identified which regulate antithrombotic and anticoagulant aptamers. Modulator efficacy can be assessed in vitro or in vivo through a coagulation test bioassay such as the activated coagulation time test, the activated partial thromboplastin test, the bleeding time test, the prothrombin time test, or the thrombin clotting time test.
  • a patient can be administered an anticoagulant nucleic acid ligand and then given the antidote when the time is appropriate to resume normal clotting action.
  • This regime is useful, for example, during cardiovascular and vascular surgery, percutaneous coronary interventions (angioplasty), orthopedic surgery, and treatment of acute myocardial infarction.
  • modulators of the present invention can bind to nucleic acid ligands that target tissue factor (TF)/factor Vila (FVIIa), factor Vina (FVUIayfactor LXa (FIXa), factor Va (F Va/factor Xa (Fxa) enzyme complexes and platelet receptors such as gp Ubllla and gp IblX and modulate the effects of the nucleic acid ligand.
  • TF tissue factor
  • FVIIa factor VUIayfactor LXa
  • FIXa factor Va
  • Fxa factor Xa
  • This invention also provides antidote controlled platelet inhibitors, antithrombotics and fibrinolytics.
  • the modulator is an oligonucleotide that binds to a Factor LXa aptamer (for example, Aptamer 9.3 or Aptamer 9.3t) that targets Coagulation Factor IXa.
  • the antidote oligonucleotide can be complementary to at least a portion of the Factor IXa aptamer.
  • the antidote 2'-O-methyl oligonucleotide can consist of the following sequence, 5'AUGGGGAGGCAGCAUUA 3 ⁇ 5'CAUGGGGAGGCAGCAUUA3', S'CAUGGGGAGGCAGCAS', 5'CAUGGGGAGGCA3',
  • the modulator is an oligonucleotide that binds to a Factor Xa aptamer (for example, Aptamer 1 lF7t) that targets Coagulation Factor Xa.
  • the antidote oligonucleotide can be complementary to at least a portion of the Factor Xa aptamer.
  • the antidote oligonucleotide may consist of the following sequences, 5'CUCGCUGGGGCUCUC3', 5'UAUUAUCUCGCUGGG3', 5 ⁇ AGAGCGGGGCCAAG3', 5'GGGCCAAGUAUUAU 3', 5' CAAGAGCGGGGCC AAG 3 ', 5'CGAGUAUUAUCUUG3' or any modification or derivative thereof wherein a desired degree of hybridization is maintained.
  • the oligonucleotide modulators include nucleic acid sequences that are substantially homologous to and that have substantially the same ability to bind nucleic acid ligands, as the oligonucleotide modulators identified herein.
  • modulators of the invention can also be used to reverse the immunosuppressive effect of nucleic acid ligands that target interleukin, for example, in patients , subject to infection.
  • the present modulators can be used to reverse the immunostimulatory effects of nucleic acid ligands that target CTLA4 in patients at risk of developing autoimmunity.
  • modulators of the invention can be used to reverse the effects of aptamers that target growth factors (e.g., PDGF or VEGF).
  • target growth factors e.g., PDGF or VEGF.
  • Such nucleic acid ligands can be used in the treatment of tumors and in the treatment of inflammatory prohferative diseases. Since growth factors play systemic roles in normal cell survival and proliferation, nucleic acid ligand treatment can result in a breakdown of healthy tissue if not tightly regulated (e.g., patients receiving nucleic acid ligands that target angiopoietin I can be subject to hemorrhaging). Modulators of the invention that target such nucleic acid ligands can be used to provide the necessary regulation.
  • Modulators of the invention can be used to reverse the effects of nucleic acid ligands that , target receptors involved in the transmission of the nerve impulse at the neuromuscular junction of skeletal muscle and/or autonomic ganglia (e.g., nicotinic acetylchoUne or nicotinic cholinergic receptors).
  • nucleic acid ligands can be made to produce muscular relaxation or paralysis during anesthesia.
  • Agents that block the activity of acetylchoUne receptors agents that engender neuromuscular blockade
  • the patients regain muscular function as soon as possible after the surgical procedure is complete to reduce complications and improve patient turnover in the operating arenas.
  • modulators of the invention that target such nucleic acid ligands can be used to provide the desired control of the activity of the neuromuscular blocker, and thus reduce the dependence on the patient's physiology to provide reversal of the neuromuscular blocking agent.
  • modulators of the invention can be used to reverse the effect of nucleic acid ligands that target small molecules, such as glucose. Hypoglycemia can be avoided in patients receiving glucose-targeted nucleic acid ligands to regulate glucose uptake using the modulators of the invention.
  • modulators can also be used to regulate the activity of nucleic acid ligands directed against members of the E2F family, certain of which are pro-proliferative, certain of which are repressive.
  • the modulators of the invention can be used to "turn on” and “turn off' such nucleic acid ligands at desired points in the cell cycle.
  • modulators of the invention can also be used to reverse the binding of nucleic acid ligands bearing radioactive or cytotoxic moieties to target tissue (e.g., neoplastic tissue) and thereby, for example, facilitate clearance of such moieties from a patient's system.
  • the modulators of the invention can be used to reverse the binding of nucleic acid ligands labeled with detectable moieties (used, for example, in imaging or cell isolation or sorting) to target cells or tissues (Hicke et al. J. Clin. Invest. 106:923 (2000); Ringquist et al, Cytometry 33:394 (1998)). This reversal can be used to expedite clearance of the detectable moiety from a patient's system.
  • Modulators of the invention can also be used in in vitro settings to enhance or inhibit the effect of a nucleic acid ligand (e.g., aptamer) on a target molecule.
  • a nucleic acid ligand e.g., aptamer
  • modulators of the invention can be used in target validation studies. Using modulators of the invention, it is possible to confirm that a response observed after inhibiting a target molecule (with a nucleic acid ligand) is due to specifically inhibiting that molecule.
  • the modulators of the invention can be formulated into pharmaceutical compositions that can include, in addition to the modulator, a pharmaceutically acceptable carrier, diluent or excipient.
  • a pharmaceutically acceptable carrier diluent or excipient.
  • the precise nature of the composition will depend, at least in part, on the nature of the modulator and the route of administration. Optimum dosing regimens can be readily established by one skilled in the art and can vary with the modulator, the patient and the effect sought.
  • the modulator can be administered IV, TM, IP, SC, or topically, as appropriate.
  • subsequent treatment can involve the administration of additional nucleic acid ligands that are either the same or different as the original nucleic acid ligand/antidote oligonucleotide pair first administered.
  • additional nucleic acid ligands that are either the same or different as the original nucleic acid ligand/antidote oligonucleotide pair first administered.
  • one optional embodiment of the invention is the identification and selection of modulators and regulators that exhibit relevant species cross-reactivity to increase usefulness of preclinical animal studies.
  • Figure 1 shows the FLXa aptamer 9.3t (SEQ ID NO: 1). All purines are 2' -hydroxyl and all pyrimidines are 2'- fluoro nucleotides.
  • Figure 2 shows the anticoagulant activity of aptamer 9.3t. Clot time increase is normalized to baseline clot time in the absence of aptamer.
  • Figure 3 shows ACT change of pigs following aptamer injection. Data are normalized to pre-injection baseline.
  • Figure 4 shows the clot time change of pigs following aptamer injection.
  • Figures 5 -5C show the in vitro inhibitory activity of cholesterol-modified aptamer, 9.3t- C.
  • Fig. 5 A Cholesterol addition has a modest effect on the affinity of aptamer 9.3t-C for FLXa. A competition binding assay is used to measure affinity of 9.3t-C for FLXa.
  • Fig. 5B In vitro anti-coagulant activity of aptamer 9.3t-C in human plasma.
  • Fig. 5C In vitro anticoagulant activity of aptamer 9.3t-C in pig plasma.
  • Figures 6A-6C show the in vivo anticoagulant activity of aptamer 9.3t-C.
  • Fig. 6A In vivo anticoagulant activity of aptamer 9.3t-C in pigs following TV bolus injection, ACT assays (dotted line is 9.3t ACT data at 0.5 mg/ml from Fig. 3).
  • Fig. 6B In vivo anticoagulant activity of aptamer 9.3t-C in pigs following IV bolus injection, APTT and PT assays (0.5 mg/kg 9.3t from Fig. 4).
  • Fig. 6C In vivo plasma concentration of 9.3t-C versus 9.3t over time foUowing bolus TV injection. Concentrations were calculated by interpolation from in vitro dose response curves of APTT assays for each aptamer.
  • Figure 8 shows the native gel analysis of binding of antidote oligonucleotide to aptamer 9.3t.
  • the ability of the antidote oligonucleotide to bind to and denature aptamer 9.3t was evaluated by native gel electrophoresis. Briefly, radiolabeled 9.3t (125 nM) was incubated at 37°C for 15 minutes with (from left to right) an 8 fold, 4 fold, 2 fold molar excess or equimolar amounts of the antidote (A.S.) or nonsense oligonucleotide (N.S.). Native 9.3t migrates faster than antidote bound 9.3t in this gel system (compare lanes 1 and 2).
  • Figures 9 A and 9B show antidote oligonucleotide reversal of anticoagulant activity of aptamer 9.3t in human plasma.
  • Fig. 9 A Change in clot time versus antidote or nonsense oligonucleotide concentration. A value of 1.0 indicates no change in clot time over the baseline value.
  • 9.3tM is a mutant version of aptamer 9.3t that has no anticoagulant activity.
  • Fig. 9B Fraction of the anticoagulant activity of aptamer 9.3t reversed versus the molar excess of antidote oligonucleotide.
  • Figure 10A- 0C show the specificity of oligonucleotide antidotes.
  • Fig. 10A Minimal secondary structure of aptamer 9.20t (SEQ UD NO: 18).
  • Fig. 10B Anticoagulant activity of 9.201
  • Fig. IOC Specificity of antidote oligonucleotide Anti DI.
  • Figure 11 shows the secondary structure of tailed aptamer 9.3t-3NT (SEQ ID NO: 19). Also shown are antidote oligonucleotides (SEQ ID NO:20 - SEQ ID NO:22).
  • Figure 12A and 12B show the activity of aptamer 9.3t-3NT.
  • Fig. 12A Competitive binding data.
  • Fig. 12B In vitro anticoagulant data.
  • Figures 13A and 13B show the in vitro reversal of the anticoagulant activity of aptamer 9.3t-3NT.
  • Fig. 13 A Reversal of anticoagulant activity vs. concentration of antidote oligonucleotide.
  • Fig. 13B Reversal of anticoagulant activity vs. molar excess of antidote over aptamer.
  • Figures 14 A and 14B show the impact of tail addition to FLXa aptamer 9.3t on the ability to reverse anticoagulant activity.
  • FIG. 18A Predicted secondary structure of aptamer 11 F7t (SEQ TD NO:23), which binds human coagulation factor Xa with a K D of -1.5 nM.
  • Fig. 18B Predicted secondary structure of a mutant version of aptamer 1 lF7t, termed 1 lF7tM (SEQ ID NO:24).
  • FIGS. 19A and 19B Aptamer 1 lF7t is a potent anticoagulant of human plasma. Varying concentrations of aptamer 1 lF7t were added to human plasma in vitro, and the clot time was then measured in a PT (Fig. 19A) or APTT assay (Fig. 19B). All data are normalized to the baseline for that day, so that a value of 1 equals no change in clot time.
  • Figure 20 Sequences of 1 lF7t to which the antidote oligos are complimentary.
  • Fig. 21 A The antidote oligonucleotides effectively reverse the activity of aptamer 1 lF7t in human plasma.
  • Fig. 21B Characterization of antidote 5-2 activity over a larger concentration range of antidote 5-2, and comparison to the antidote activity of a scrambled sequence version of antidote 5-2, 5-2 ser.
  • FIG 22 Kinetics of antidote activity in human plasma.
  • Aptamer 1 lF7t was added to human plasma in vitro at a final concentration of 125 nM, and allowed to incubate for 5 minutes at 37°C.
  • Antidote oligonucleotide 5-2 at a 1:1 or 5:1 molar excess or no antidote were then added, and the clotting activity of the plasma determined by measuring the clotting time in a PT assay at the times indicated following antidote addition.
  • the % residual anticoagulant activity remaining was calculated by comparing the difference over baseline between the clotting time in the presence of antidote to the difference over baseline in the absence of antidote at each time point.
  • the data collected at a 5:1 molar excess of antidote 5-2 to aptamer 1 lF7t is not shown, as reversal of the anticoagulant activity was complete at the first time point (1 minute).
  • Figure 23 Duration of antidote activity in vitro.
  • the duration of the inactivation of the anticoagulant activity of aptamer HF7t by antidote oligonucleotide 5-2 was measured in vitro in human plasma. Briefly, 1 lF7t was added to human plasma to a final concentration of 125 nM and allowed to incubate for 5 minutes.
  • Antidote oligonucleotide 5-2 was then added at a 4 fold molar excess, or in a parallel experiment buffer alone was added in place of the antidote oligo, and the clotting time was measured in an a PT assay at various time points following antidote addition.
  • FIG. 24 Aptamers 9.3t and 1 lF7t and their respective antidotes function independent of each other in human plasma.
  • Aptl 9.3t (30 nM)
  • Apt2 llF7t (100 nM)
  • ADl AO 5-2c (300 nM)
  • AD2 AO5-2 (500 nM)- final concentrations in plasma in Q.
  • Aptamers were added to human plasma at 37°C as indicated, and allowed to incubate for 5 minutes. Antidotes were then added as indicated, and the clotting activity was measured 10 minutes after antidote addition in APTT assays.
  • FIGS. 25 A - 25F Antidote-controlled anticoagulation of plasma from patients with heparin-induced thrombocytopenia.
  • Figs. 25A-25C The activity of aptamer Peg-9.3t and antidote 5-2 were tested in plasma from hemodialysis-dependent patients diagnosed with HIT.
  • Fig. 25D-25F The activity of aptamer Peg-9.3t and antidote 5-2 were tested in plasma from patients suffering from thromboembolic complications of HIT.
  • Plasma samples were treated as indicated: aptamer, 125 nM Peg-9.3t; antidote, 1.25 ⁇ M AO 5-2; mutant aptamer, 125 nM 9.3tM. Experiments were performed as described in Example 2, Figure 9. Data is reported in seconds (s) and is the average ⁇ range of duplicate measurements.
  • FIGS 26A and B Antidote-controlled anticoagulation of plasma from patients with heparin-induced thrombocytopenia.
  • the activity of aptamer 1 lF7t and antidote 5-2 were tested in plasma from a hemodialysis-dependent patient diagnosed with HIT and from a patient suffering from thromboembolic complications of HIT.
  • plasma samples were treated as indicated: aptamer, 250 nM 1 lF7t; antidote, 1.0 ⁇ M AO 5-2; mutant aptamer, 250 nM 9.3tM. Experiments were performed as described in Example 2, Figure 9.
  • aptamer 125 nM 1 lF7t
  • antidote 250 nM AO 5-2
  • mutant aptamer 125 nM 9.3tM.
  • Data is reported in seconds (s) and is the average ⁇ range of duplicate measurements.
  • the present invention relates generally to modulators of pharmacological agents, including therapeutic and diagnostic agents.
  • the invention further relates to methods of enhancing or inhibiting the efficacy of pharmacological agents by administering modulators of the invention to a subject (e.g., a human) in need thereof.
  • the invention relates to methods of using modulators of the invention to assess the activity of nucleic acid ligands, in vivo and in vitro.
  • the present invention relates to a method of modulating the activity of a nucleic acid ligand, for example, by altering its conformation and thus its function.
  • the modulator can be contacted with the targeted nucleic acid ligand under conditions such that it binds to the nucleic acid ligand and modifies the interaction between the nucleic acid ligand and its target molecule. Modification of that interaction can result from modification of the nucleic acid ligand structure as a result of binding by the modulator.
  • the modulator can bind the free nucleic acid ligand and/or the nucleic acid ligand bound to its target molecule.
  • Modulators of the invention can be designed so as to bind any particular nucleic acid ligand with a high degree of specificity and a desired degree of affinity. Modulators can be also be designed so that, upon binding, the structure of the nucleic acid ligand is modified to either a more or less active form. For example, the modulator can be designed so that upon binding to the targeted nucleic acid ligand, the three-dimensional structure of that nucleic acid ligand is altered such that the nucleic acid ligand can no longer bind to its target molecule or binds to its target molecule with less affinity.
  • the modulator can be designed so that, upon binding, the three dimensional structure of the nucleic acid ligand is altered so that the affinity of the nucleic acid ligand for its target molecule is enhanced. That is, the modulator can be designed so that, upon binding, a structural motif is produced in the nucleic acid ligand so that the nucleic acid ligand can bind to its target molecule.
  • the modulator is an oligonucleotide.
  • the oligonucleotide can be a sequence that is complementary to at least a portion of the nucleic acid ligand.
  • the modulator is a ribozyme or DNAzyme that targets the nucleic acid ligand.
  • the modulator can be, for example, a peptide nucleic acid or morpholino nucleic acid that includes a sequence that is complementary to or hybridizes with at least a portion of the nucleic acid ligand.
  • a modulator is specifically hybridizable with the nucleic acid ligand when binding of the modulator to the nucleic acid ligand sufficiently interferes with the normal function of the nucleic acid ligand to cause a change in the biological activity of the nucleic acid ligand, under physiological conditions.
  • the modulator is nucleic acid binding peptide, polypeptide or protein that binds to or otherwise interacts with the nucleic acid ligand.
  • the modulator is an oligosaccharide that binds the nucleic acid ligand.
  • the modulator is an aminoglycoside.
  • the modulator is a small organic molecule (i.e., a molecule that can be synthetic or naturally occurring that is not otherwise found in vivo and typically has a molecular weight of less than 1000).
  • the present invention also includes methods for identifying modulators of nucleic acid ligands.
  • the binding of a modulator to a nucleic acid is determined by any assay that measures binding affinity, such as a gel shift assay.
  • the binding of a modulator to a nucleic acid ligand is determined by a Biacore assay. Other exemplary assays are described below.
  • the binding or interaction of the modulator with the nucleic acid ligand is measured by evaluating the effect of the hgand with and without the regulator under appropriate biological conditions.
  • modulators can be identified that modify the antithrombotic or anticoagulant activity of nucleic acid ligand in vitro or in vivo through a coagulation test bioassay such as an activated coagulation time test, the activated partial thromboplastin test, the bleeding time test, the prothrombin time test, or the thrombin clotting time test.
  • the present invention further includes the use of such modulators in a variety of indications whereby control of nucleic acid ligand activity is desired.
  • the modulators may act to inhibit nucleic acid ligand activity as an antidote to reverse the actions of the nucleic acid ligand.
  • the invention provides methods of using modulators of the invention to assess the activity of nucleic acid ligands.
  • the invention is also directed to methods of enhancing or inhibiting the efficacy of nucleic acid ligands by administering modulators of the invention to human or non-human mammals.
  • modulators of the invention can also be used to reverse the immunosuppressive effect of nucleic acid ligands that target interleukin, for example, in patients subject to infection.
  • the present modulators can be used to reverse the immunostimulatory effects of nucleic acid ligands that target CTLA4 in patients at risk of developing autoimmunity.
  • modulators of the invention can be used to reverse the effects of nucleic acid ligands that target growth factors (e.g., PDGF or VEGF).
  • nucleic acid ligands can be used in the treatment of tumors and in the treatment of inflammatory prohferative diseases. Since growth factors play systemic roles in normal cell survival and proliferation, nucleic acid ligand treatment can result in a breakdown of healthy tissue if not tightly regulated (e.g., patients receiving nucleic acid ligands that target angiopoietin I can be subject to hemorrhaging). Modulators of the invention that target such nucleic acid ligands can be used to provide the necessary regulation.
  • Modulators of the invention can be used to reverse the effects of nucleic acid ligands that target receptors involved in the transmission of the nerve impulse at the neuromuscular junction of skeletal muscle and/or autonomic ganglia (e.g., nicotinic acetylchoUne or nicotinic cholinergic receptors).
  • nucleic acid ligands can be made to produce muscular relaxation or paralysis during anesthesia.
  • Agents that block the activity of acetylchoUne receptors agents that engender neuromuscular blockade
  • the patients regain muscular function as soon as possible after the surgical procedure is complete to reduce complications and improve patient turnover in the operating arenas.
  • modulators of the invention that target such nucleic acid ligands can be used to provide the desired control of the activity of the neuromuscular blocker, and thus reduce the dependence on the patient's physiology to provide reversal of the neuromuscular blocking agent.
  • modulators of the invention can be used to reverse the effect of nucleic acid ligands that target small molecules, such as glucose. Hypoglycemia can be avoided in patients receiving glucose-targeted nucleic acid ligands to regulate glucose uptake using the modulators of the invention.
  • modulators can also be used to regulate the activity of nucleic acid ligands directed against members of the E2F family, certain of which are pro-proliferative, certain of which are repressive.
  • the modulators of the invention can be used to "turn on” and “turn off' such nucleic acid ligands at desired points in the cell cycle.
  • modulators of the invention can also be used to reverse the binding of nucleic acid ligands bearing radioactive or cytotoxic moities to target tissue (e.g., neoplastic tissue) and thereby, for example, facilitate clearance of such moeity's from a patient's system.
  • the modulators of the invention can be used to reverse the binding of nucleic acid ligands labeled with detectable moieties (used, for example, in imaging or cell isolation or sorting) to target cells or tissues (Hicke et al. J. Clin. Invest. 106:923 (2000); Ringquist et al, Cytometry 33:394 (1998)). This reversal can be used to expedite clearance of the detectable moiety from a patient's system.
  • Modulators of the invention can also be used in in vitro settings to enhance or inhibit the effect of a nucleic acid ligand (e.g., aptamer) on a target molecule.
  • a nucleic acid ligand e.g., aptamer
  • modulators of the invention can be used in target validation studies. Using modulators of the invention, it is possible to confirm that a response observed after inhibiting a target molecule (with a nucleic acid ligand) is due to specifically inhibiting that molecule.
  • the modulators of the invention can be formulated into pharmaceutical compositions that can include, in addition to the modulator, a pharmaceutically acceptable carrier, diluent or excipient.
  • a pharmaceutically acceptable carrier diluent or excipient.
  • the precise nature of the composition will depend, at least in part, on the nature of the modulator and the route of administration. Optimum dosing regimens can be readily established by one skilled in the art and can vary with the modulator, the patient and the effect sought. Generally, the modulator is administered IV, IM, IP, SC, orally or topically, as appropriate.
  • the nucleic acid ligand or its regulator can be covalently attached to a lipophilic compound such as cholesterol, dialkyl glycerol, diacyl glycerol, or a non- immunogenic, high molecular weight compound or polymer such as polyethylene glycol (PEG).
  • a lipophilic compound such as cholesterol, dialkyl glycerol, diacyl glycerol, or a non- immunogenic, high molecular weight compound or polymer such as polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the nucleic acid ligand or the modulator can be comprised for example, of a nucleic acid or PNA or MNA encapsulated inside a Hposome, and the enhanced intracellular uptake is seen over the un-complexed oligonucleotide or modulator.
  • the lipophilic compound or non-immunogenic, high molecular weight compound can be covalently bonded or associated through non-covalent interactions with Hgand or modulator(s).
  • the lipophilic compound is cholesterol, dialkyl glycerol, diacyl glycerol, or the non- immunogenic, high molecular weight compound is PEG, a covalent association with the oligonucleotide modulator (s) is preferred.
  • the lipophilic compound is a cationic Hposome or where the oligonucleotide modulators are encapsulated within the Hposome, a non-covalent association with the oligonucleotide modulator (s) is preferred.
  • the lipophilic compound or non-immunogenic, high molecular weight compound may be covalently bound to a variety of positions on the oligonucleotide modulator, such as to an exocyclic amino group on the base, the 5-position of a pyrimidine nucleotide, the 8-position of a purine nucleotide, the hydroxyl group of the phosphate, or a hydroxyl group or other group at the 5' or 3' terminus of the oligonucleotide modulator. Preferably, however, it is bonded to the 5' or 3' hydroxyl group thereof.
  • Attachment of the oligonucleotide modulator to other components of the complex can be done directly or with the utilization of linkers or spacers.
  • the lipophilic compound or non-immunogenic, high molecular weight compound can associate through non-covalent interactions with the oligonucleotide modulator(s).
  • the oligonucleotide modulator is encapsulated within the internal compartment of the Hpophilic compound.
  • the oligonucleotide modulator associates with the lipophilic compound through electrostatic interactions.
  • a cationic Hposome can associate with an anionic oligonucleotide modulator.
  • Another example of a non-covalent interaction through ionic attractive forces is one in which a portion of the oligonucleotide modulator hybridizes through Watson-Crick base-pairing or triple helix base pairing to an oligonucleotide which is associated with a lipophilic compound or non- immunogenic, high molecular weight compound.
  • binding activity and "binding affinity' are meant to refer to the tendency of a ligand molecule to bind or not to bind to a target.
  • the energy of said interactions are significant in "binding activity” and "binding affinity” because they define the necessary concentrations of interacting partners, the rates at which these partners are capable of associating, and the relative concentrations of bound and free molecules-in a solution.
  • the energetics are characterized through, among other ways, the determination of a dissociation constant, Kd.
  • the Kd is established using a double-filter nitroceUulose filter binding assay such as that disclosed by Wong and Lohman, 1993, Proc. Natl Acad. Sci. 'USA 90, 5428-5432.
  • Specifically binding oHgonucleotides are oligonucleotides having specific binding regions that are capable of forming complexes with an intended target molecule.
  • the specificity of the binding is defined in terms of the comparative dissociation constants (Kd) of a modulator of a nucleic acid Hgand as compared to the dissociation constant with respect to other materials in the environment or unrelated molecules in general.
  • Kd comparative dissociation constants
  • the K for a modulator of a nucleic acid ligand can be 2-fold, preferably 5-fold, more preferably 10-fold less than the Kd with respect to the modulator and the unrelated material or accompanying material in the environment.
  • the Kd will be 50-fold less, more preferably 100-fold less, and more preferably 200-fold less.
  • d can be determined directly by well-known methods, and can be computed even for complex mixtures by methods such as those, for example, set forth in Caceci, M., et al., Byte (1984) 9:340-362. It has been observed, however, that for some small oligonucleotides, direct determination of Kd is difficult, and can lead to misleadingly, high results. Under these circumstances, a competitive binding assay for the target molecule or other candidate substance can be conducted with respect to substances known to bind the target or candidate. The value of the concentration at which 50% inhibition occurs (Ki) is, under ideal conditions, equivalent to K d .
  • K is less than Kd.
  • determination of Ki sets a maximal value for the value of Kd.
  • measurement of Ki can conveniently be substituted to provide an upper limit for K .
  • a K value can also be used to confirm that a modulator binds a nucleic acid ligand.
  • the present invention it is preferred to exclude from the categories of unrelated materials and materials accompanying the target in the target's environment those materials which are sufficiently related to the target to be immunologically cross-reactive therewith.
  • immunologically cross-reactive is meant that antibodies raised with respect to the target cross- react under standard assay conditions with the candidate material.
  • the binding affinities of the antibodies for cross-reactive materials as compared to targets should be in the range of 5-fold to 100-fold, generally about 10-fold.
  • hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases.
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides.
  • oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position.
  • the oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other.
  • “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target.
  • sequence of compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.
  • a compound is specifically hybridizable when binding of the compound to the target nucleic acid molecule interferes with the normal function of the target nucleic acid to cause a change in utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.
  • Oligonucleotides include RNA or DNA sequences or mixtures or analogs thereof, of more than one nucleotide in either single chain or duplex form and specifically includes short sequences such as dimers and trimers, in either single chain or duplex form, which can be intermediates in the production of the specifically binding oligonucleotides.
  • “Modified” forms used in candidate pools contain at least one non-native residue.
  • RNA analog is meant to refer to a polymeric molecule which can contain one or more nucleotides that have a nonhydrogen substituent other than a hydroxyl group in the 2' -position, and for example, can contains at least one of the following: 2'-deoxy, 2'-halo (including 2'-fluoro), 2'-amino (preferably not substituted or mono- or disubstituted), 2'-mono-, di- ortri-halomethyl, 2'-O-alkyl (including 2'-O-methyl or O-ethyl), 2'-O-halo-substituted alkyl, 2' -alkyl, azido, phosphorothioate, sulfhydryl, methylphosphonate, fluorescein, rhodamine, pyrene, biotin, xanthine, hypoxanthine, 2,6-diamino purine, 2-hydroxy-6-mercaptopurine and pyr
  • a "target” or “target molecule” refers to a biomolecule that is the focus of a. therapeutic drug strategy or diagnostic assay, including, without limitation, enzymes, enzyme inhibitors, hormones, glycoproteins, lipids, phospholipids, nucleic acids, intracellular, extracellular, and cell surface proteins, peptides, carbohydrates, including glycosaminoglycans, lipids, including glycolipids and certain oligonucleotides, and generally, any biomolecule capable of turning a biochemical pathway on or off or modulating it, or which is involved in a predictable biological response.
  • Targets can be free in solution, like thrombin, or associated with cells or viruses, as in receptors or envelope proteins. Any molecule that is of sufficient size to be specifically recognized by a nucleic acid ligand can be used as the target.
  • membrane structures, receptors, organelles, and the like can be used as the complexation targets.
  • RNA aptamer is an aptamer comprising ribonucleoside units. "RNA aptamer” is also meant to encompass RNA analogs as defined herein above.
  • coagulation factor aptamer is meant to refer to a single- or double-stranded nucleic acid that binds a coagulation factor and modulates its function.
  • coagulation factor is meant to refer to a factor that acts in either or both of the intrinsic and the extrinsic coagulation cascade.
  • Consensus sequence refers to a nucleotide sequence or region (which might or might not be made up of contiguous nucleotides) that is found in one or more regions of at least two nucleic acid sequences.
  • a consensus sequence can be as short as three nucleotides long. It also can be made up of one or more noncontiguous sequences, with nucleotide sequences or polymers of up to hundreds of bases long interspersed between the consensus sequences. Consensus sequences can be identified by sequence comparisons between individual nucleic acid species, which comparisons can be aided by computer programs and other, tools for modeling secondary and tertiary structure from sequence information. Generally, the consensus sequence will contain at least about 3 to 20 nucleotides, more commonly from 6 to 10 nucleotides.
  • cardiovascular disease and “cardiovascular diseases” are meant to refer to any cardiovascular disease as would be understood by one of ordinary skill in the art.
  • cardiovascular diseases include, but are not limited to, atherosclerosis, thrombophilia, embolisms, cardiac infarction (e.g., myocardial infarction), thromboses, angina, stroke, septic shock, hypertension, hyper-cholesterolemia, restenosis and diabetes.
  • optically active and racemic forms may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism.
  • the present invention encompasses racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein.
  • the optically active forms can be prepared by, for example, resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase or by enzymatic resolution.
  • the compounds are used in naturally occurring forms. However, alternatively, the compounds can be used in a non-naturally occurring form.
  • a “Nucleic Acid Ligand” (sometimes also referred to as an "aptamer”) as used herein is a non-naturally occurring nucleic acid having a desirable action on a target.
  • a desirable action includes, but is not limited to, binding of the target, catalytically changing the target, reacting with the target in a way which modifies/alters the target or the functional activity of the target, covalently attaching to the target as in a suicide inhibitor, or facilitating the reaction between the target and another molecule.
  • the action is specific binding affinity for a target molecule, such target molecule being a three dimensional chemical structure, wherein the nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule.
  • nucleic acid ligands are identified using the SELEX methodology.
  • Nucleic acid ligands includes nucleic acids that are identified from a candidate mixture of nucleic acids, wherein the nucleic acid ligand being a ligand of a given target by the method comprising a) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; b) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; and c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids.
  • nucleic acid ligand or aptamer denotes both singular and plural sequences of nucleic acids which are capable of binding to a protein or other molecule, and thereby disturbing the protein's or other molecule's function.
  • Nucleic acid ligands can be made with nucleotides bearing D or L stereochemistry, or a mixture thereof. Naturally occurring nucleosides are in the D configuration. Aptamers have been made from L nucleotides, and are called L-aptamers. Nucleic acid ligands used for therapeutic purposes are typically in the D configuration, but can exhibit any configuration that provides the desired effect. Typically, when nucleic acid ligands comprising L-nucleotides are the target of an oligonucleotide modulator, the modulator also comprises L-nucleotides (see, for example, USP 5,780,221).
  • the nucleic acid ligands preferably comprise about 10 to about 100 nucleotides, preferably about 15 to about 40 nucleotides, more preferably about 20 to about 40 nucleotides, in that oligonucleotides of a length that falls within these ranges are readily prepared by conventional techniques.
  • aptamers or oligonucleotide modulators can comprise a minimum of approximately 6 nucleotides, preferably 10, and more preferably 14 or 15 nucleotides, that are necessary to effect specific binding.
  • the only apparent limitations on the binding specificity of the target/oligonucleotide couples of the invention concern sufficient sequence to be distinctive in the binding oligonucleotide and sufficient binding capacity of the target substance to obtain the necessary interaction.
  • Aptamers of binding regions containing sequences shorter than 10, e.g., 6-mers, are feasible if the appropriate interaction can be obtained in the context of the environment in which the target is placed. Thus, if there is little interference by other materials, less specificity and less strength of binding can be required.
  • nucleic acid ligands and methods for their production and use are described, for example, in the following U.S. patents. Any of the nucleic acid ligands described in the patents listed below or other patents, or any nucleic acid ligands described in publications as well as other desired nucleic acid ligands used in medical therapy can be modulated or regulated according to the present invention.
  • U.S. Patent No. 6,387,620 entitled Transcription-free selex;
  • VEGF Vascular endothelial growth factor
  • U.S. Patent No. 6,147,204 entitled Nucleic acid ligand complexes
  • U.S. Patent No. 6,140,490 entitled High affinity nucleic acid ligands of complement system proteins
  • U.S. Patent No. 6,127,119 entitled Nucleic acid ligands of tissue target
  • U.S. Patent No. 6,124,449 entitled High affinity TGF ⁇ nucleic acid ligands and inhibitors
  • U.S. Patent No. 6,114,120 entitled Systematic evolution of ligands by exponential enrichment: tissue selex
  • Patent No.5,837,456 entitled High affinity oligonucleotide ligands to chorionic gonadotropin hormone and related glycoprotein hormones
  • U.S. Patent No. 5,834,199 entitled Methods of identifying transition metal complexes that selectively cleave regulatory elements of mRNA and uses thereof
  • U.S. Patent No.5,817,785 entitled Methods of producing nucleic acid ligands
  • U.S. Patent No.5,811,533 entitled High- affinity oligonucleotide ligands to vascular endothelial growth factor (VEGF)
  • U.S. Patent No.5,795,721 entitled High affinity nucleic acid ligands of ICP4
  • Patent No.5,766,853 entitled Method for identification of high affinity nucleic acid ligands to selectins; U.S. Patent No.5,763,595, entitled Systematic evolution of ligands by exponential emichment: Chemi-SELEX; U.S. Patent No. 5,763,566, entitled Systematic evolution of ligands by exponential enrichment: tissue SELEX; U.S. Patent No.5,763,177, entitled Systematic evolution of ligands by exponential enrichment: photoselection of nucleic acid ligands and solution selex; U.S. Patent No.5,763,173, entitled Nucleic acid ligand inhibitors to DNA polymerases; U.S.
  • Patent No.5,756,287 entitled High affinity HTV integrase inhibitors
  • U.S. Patent No. 5,750,342 entitled Nucleic acid ligands of tissue target
  • U.S. Patent No.5,734,034 entitled Nucleic acid ligand inhibitors of human neutrophil elastase
  • U.S. Patent No.5, 731,424 entitled High affinity TGF ⁇ nucleic acid ligands and inhibitors
  • U.S. Patent No.5,731,144 entitled High affinity TGF ⁇ nucleic acid ligands
  • U.S. Patent No. 5,726,017 entitled High affinity HTV-1 gag nucleic acid ligands
  • Patent No.5,648,214 entitled High- affinity oligonucleotide ligands to the tachykinin substance P
  • U.S. Patent No.5,641,629 entitled Spectroscopically detectable nucleic acid ligands
  • U.S. Patent No.5,639,868 entitled High- affinity RNA ligands for basic fibroblast growth factor
  • U.S. Patent No. 5,637,682 entitled High-affinity oligonucleotide ligands to the tachykinin substance P
  • Patent No.5,637,459 entitled Systematic evolution of ligands by exponential enrichment: chimeric selex; U.S. Patent No.5,635,615, entitled High affinity HTV nucleocapsid nucleic acid ligands; U.S. Patent No.5,629,155, entitled High-affinity oligonucleotide ligands to immunoglobulin E (IgE); U.S. Patent No. 5,622,828, entitled High- affinity oligonucleotide ligands to secretory phospholipase A2 (sPLAsub.2); U.S. Patent No. 5,595,877, entitled Methods of producing nucleic acid ligands; U.S.
  • VEGF Vascular endothelial growth factor
  • Patent 5,849,890 entitled High affinity oligonucleotide ligands to chorionic gonadotropin hormone and related glycoprotein hormones
  • U.S. Patent 5,849,479 entitled High-affinity oligonucleotide ligands to vascular endothelial growth factor (VEGF)
  • U.S. Patent 5,846,713, entitled High affinity HKGF nucleic acid ligands and inhibitors
  • U.S. Patent 5,843,732 entitled Method and apparatus for determining consensus secondary structures for nucleic acid sequences
  • Patent 5,840,867 entitled Aptamer analogs specific for biomoiecules
  • U.S. Patent 5,840,580 entitled Phenotypic characterization of the hematopoietic stem cell
  • U.S. Patent 5,834,199 entitled Methods of identifying transition metal complexes that selectively cleave regulatory elements of mRNA and uses thereof;
  • Patent 5,834,184 entitled In vivo selection of RNA-binding peptides
  • U.S. Patent 5,817,785 entitled Methods of producing nucleic acid ligands
  • U.S. Patent 5,804,390 entitled Use of nuclear magnetic resonance to identify ligands to target biomoiecules
  • Patent 5,789,160 entitled Parallel selex
  • U.S. Patent 5,789,157 entitled Systematic evolution of ligands by exponential enrichment: tissue selex
  • U.S. Patent 5,786,462 entitled High affinity ssDNA ligands of HTV-1 reverse transcriptase
  • U.S. Patent 5,786,203 entitled Isolated nucleic acid encoding corticotropin-releasing factor.sub.2 receptors
  • U.S. Patent 5,786,145 entitled Oligonucleotide competitors for binding of HTV RRE to REV protein and assays for screening inhibitors of this binding
  • Patent 5,780,610 entitled Reduction of nonspecific hybridization by using novel base-pairing schemes
  • U.S. Patent 5,766,853 entitled Method for identification of high affinity nucleic acid ligands to selectins
  • Patent 5,750,342 entitled Nucleic acid ligands of tissue target; U.S. Patent 5,739,305, entitled Nucleotide-directed assembly of bimolecular and multimolecular drugs and devices; U.S. Patent 5,734,034, entitled Nucleic acid ligand inhibitors of human neutrophil elastase; U.S. Patent 5,733,732, entitled Methods for detecting primary adhalinopathy; U.S. Patent 5,731,424, entitled High affinity TGF.beta. nucleic acid ligands and inhibitors; U.S. Patent 5,731,144, entitled High affinity TGF.beta. nucleic acid ligands; U.S.
  • Patent 5,726,017 entitled High affinity HTV-1 gag nucleic acid ligands
  • U.S. Patent 5,726,014 entitled Screening assay for the detection of DNA-binding molecules
  • U.S. Patent 5,723,594 entitled High affinity PDGF nucleic acid ligands
  • U.S. Patent No. 5,723,592 entitled Parallel selex
  • U.S. Patent No. 5,723,289 entitled Parallel selex
  • U.S. Patent No. 5,712,375 entitled Systematic evolution of ligands by exponential enrichment: tissue selex
  • Modulators i.e., regulators of the invention include any pharmaceutically acceptable agent that can bind a nucleic acid ligand and modify the interaction between that ligand and its target molecule (e.g., by modifying the structure of the aptamer) in a desired manner, or which degrades, metabolizes, cleaves or otherwise chemically alters the nucleic acid ligand to modify its biological effect.
  • modulators include (A) oligonucleotides or analogues thereof that are complementary to at least a portion of the aptamer sequence (including ribozymes or DNAzymes or, for example, peptide nucleic acids (PNAs), mopholino nucleic acids (MNAs), or Locked Nucleic Acids (LNAs)), (B) nucleic acid binding peptides, polypeptides or proteins (including nucleic acid binding tripeptides (see, generally, Hwang et al., Proc. Natl. Acad. Sci. USA 96:12997 (1999)), (C) oligosaccharides (e.g.
  • the modulator itself is an aptamer.
  • a nucleic acid ligand is first generated that binds to the desired therapeutic target.
  • a second aptamer that binds to the first aptamer is generated using the SELEX process described herein or other process, and modulates the interaction between the therapeutic aptamer and the target.
  • the nucleic acid ligand which binds to the target is a PNA, MNA, LNA or PCO and the modulator is an aptamer.
  • the nucleic acid ligand which binds to the target is a PNA, MNA, LNA or PCO, and the modulator is an MNA.
  • the nucleic acid ligand which binds to the target is a PNA, MNA, LNA or PCO
  • the modulator is an LNA.
  • the nucleic acid ligand which binds to the target is a PNA, MNA, LNA or PCO
  • the modulator is a PCO.
  • the modulator of the invention is an oligonucleotide that comprises a sequence complementary to at least a portion of the targeted nucleic acid ligand sequence.
  • the modulator oligonucleotide can comprise a sequence complementary to 6-25 nucleotides of the targeted nucleic acid ligand, typically, 8-20 nucleotides, more typically, 10-15 nucleotides.
  • the modulator oligonucleotide is complementary to 6-25 consecutive nucleotides of the nucleic acid ligand, or 8-20 or 10-15 consecutive nucleotides.
  • the length of the modulator oligonucleotide can be readily optimized taking into account the targeted nucleic acid ligand and the effect sought.
  • the modulator oligonucleotide is 5-80 nucleotides in length, more typically, 10-30 and most typically 15-20 nucleotides (e.g., 15-17).
  • the oligonucleotide can be made with nucleotides bearing D or L stereochemistry, or a mixture thereof. Naturally occurring nucleosides are in the D configuration.
  • duplexes by binding of complementary pairs of short oligonucleotides is a fairly rapid reaction with second order association rate constants generally between lxl 0 6 and 3xl0 5 M 1 s 1 .
  • the initial phase of the binding reaction involves formation of a two to three base pair duplex, termed "nucleation". After nucleation, pairing proceeds through the entire length of the complementary sequence at a very fast rate ⁇ approximately 10 6 bases per second at 20°C.
  • the modulatory effect on a nucleic acid ligand by formation of a duplex with a complimentary oligonucleotide is rapid. Stability of short duplexes is highly dependent on the length and base-composition of the duplex.
  • thermodynamic parameters for formation of short nucleic acid duplexes have been rigorously measured, resulting in nearest- neighbor rules for all possible base pairs such that accurate predictions of the free energy, T m and thus half-life of a given oligoribonucleotide duplex can be calculated (e.g., Xia et al, Biochem. 37:14719 (1998) (see also Eguchi et al. Antigensis RNA, Annu. Rev. Biochem. 60:631 (1991)).
  • Oligonucleotide modulators of the invention are advantageously targeted at single- stranded regions of the nucleic acid ligand. This facilitates nucleation and, therefore, the rate of nucleic acid ligand activity modulation, and also, generally leads to intermolecular duplexes that contain more base pairs than the targeted nucleic acid ligand.
  • Various strategies can be used to determine the optimal site for oligonucleotide binding to a targeted nucleic acid ligand.
  • An empirical strategy can be used in which complimentary oligonucleotides are "walked" around the aptamer. A walking experiment can involve two experiments performed sequentially.
  • a new candidate mixture can be produced in which each of the members of the candidate mixture has a fixed nucleic acid- region that corresponds to a oligonucleotide modulator of interest.
  • Each member of the candidate mixture also contains a randomized region of sequences. According to this method it is possible to identify what are referred to as "extended" nucleic acid ligands, which contain regions that can bind to more than one binding domain of an nucleic acid ligand.
  • 2'-O-methyl oligonucleotides e.g., 2'-O-methyl oligonucleotides
  • about 15 nucleotides in length can be used that are staggered by about 5 nucleotides on the aptamer (e.g., oligonucleotides complementary to nucleotides 1-15, 6-20, 11-25, etc. of aptamer 9.3t.
  • An empirical strategy can be particularly effective because the impact of the tertiary structure of the aptamer on the efficiency of hybridization can be difficult to predict.
  • Assays described in the Examples that follow can be used to assess the ability of the different oligonucleotides to hybridize to a specific nucleic acid ligand, with particular emphasis on the molar excess of the oligonucleotide required to achieve complete binding of the nucleic acid ligand.
  • the ability of the different oligonucleotide modulators to increase the rate of dissociation of the nucleic acid ligand from, or association of the nucleic acid ligand with, its target molecule can also be determined by conducting standard kinetic studies using, for example, BIACORE assays.
  • Oligonucleotide modulators can be selected such that a 5-50 fold molar excess of oligonucleotide, or less, is required to modify the interaction between the nucleic acid Hgand and its target molecule in the desired manner.
  • the targeted nucleic acid ligand can be modified so as to include a single- stranded tail (3' or 5') in order to promote association with an oligonucleotide modulator.
  • Suitable tails can comprise 1 to 20 nucleotides, preferably, 1-10 nucleotides, more preferably, 1- 5 nucleotides and, most preferably, 3-5 nucleotides (e.g., modified nucleotides such as 2'-O- methyl sequences).
  • Tailed nucleic acid ligands can be tested in binding and bioassays (e.g., as described in the Examples that follow) to verify that addition of the single-stranded tail does not disrupt the active structure of the nucleic acid ligand.
  • a series of oligonucleotides that can form, for example, 1, 3 or 5 base pairs with the tail sequence can be designed and tested for their ability to associate with the tailed aptamer alone, as well as their ability to increase the rate of dissociation of the nucleic acid ligand from, or association of the aptamer with, its target molecule.
  • Scrambled sequence controls can be employed to verify that the effects are due to duplex formation and not non-specific effects.
  • the native gel assay described in the Examples that follow can be used to measure the dissociation association rate of an oligonucleotide modulator from/with a target aptamer.
  • the present invention provides modulators that specifically and rapidly reverse the anticoagulant and antithrombotic effects of nucleic acid ligands that target components of the coagulation pathway, particularly nucleic acid Hgand antagonists of the tissue factor (TF)/factor Vila (FVIIa), factor VHIa (FVffla)/factor LXa (FLXa), factor Va (FVa factor Xa (Fxa) enzyme complexes and platelet receptors such as gpIIblUa, gpIblX, gpVI, factors involved in promoting platelet activation such as Gas6, factors involved in promoting or maintaining fibrin clot formation such as PAI-1 (plasminogen activator inhibitor 1) or coagulation factor XTfla (FXTJIa), and additional factors involved in promoting or preventing fibrin clot formation such as ATIJJ (anti-thrombin HI), thrombin or coagulation factor XIa (FXIa).
  • TF tissue factor
  • modulators of nucleic acid ligand activity are nucleic acids selected from the group consisting of, but not limited to the following sequences; 5'AUGGGGAGGCAGCAUUA 3' (SEQ ID NO:25), 5'CAUGGGGAGGCAGCAUUA3' (SEQ ID NO:26), 5'CAUGGGGAGGCAGCA3' (SEQ ID NO:27), 5'CAUGGGGAGGCA3' (SEQ TO NO:28), 5'GCAUUACGCGGUAUAGUCCCCUA3 ' (SEQ ID NO:29),
  • the inhibitor of a nucleic acid ligand to Factor LXa of the present invention can be an antisense oligonucleotide.
  • the antisense oligonucleotide hybridizes to the nucleic acid ligand in vivo and blocks the binding of the nucleic acid Hgand to factor LXa.
  • the oligonucleotide modulators of the invention comprise a sequence complementary to at least a portion of a nucleic acid ligand. However, absolute complementarity is not required.
  • the oligonucleotide can be at least 5 or at least 10 nucleotides, at least 15 or 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
  • the oligonucleotides of the invention can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single- stranded.
  • Antisense techniques are discussed for example, in Okano, L, Neurochein. 56:560 (1991);Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton,FL (1988). The methods are based on binding of a polynucleotide to a complementary DNA or RNA.
  • Oligonucleotides of the invention can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
  • the oligonucleotide can include other appended groups.
  • the oligonucleotide can be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
  • the oligonucleotide can comprise at least one modified base moiety which is selected from the group including, but not limited to, 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5-(carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine,N6- isopentenyladenine, 1 -methylguanine, 1-methylinosine, 2,2-dimethylguanine,2-methyladenine, 2-methylguanine, 3-inethylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2 ⁇ -thiouracil, beta-D-mannos
  • An oligonucleotide modulator of the invention can also comprise at least one modified sugar moiety selected from the group including, but not Hmited to, arabinose, 2-fluoroarabinose, xylose, and hexose.
  • an oligonucleotide modulator can comprise at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphorodiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • Oligonucleotide modulators can be prepared that have desired characteristics, such as improved in vivo stability and/or improved delivery characteristics. Examples of such modifications include chemical substitutions at the sugar and/or backbone and/or base positions. Oligonucleotide modulators can contain nucleotide derivatives modified at the 5- and 2' positions of pyrimidines, for example, nucleotides can be modified with 2' amino, 2'-fluoro and/or 2'-O- methyl. Modifications of the modulating oligonucleotides of the invention can include those that provide other chemical groups that incorporate additional charge, polarization, hydrophobicity, hydrogen bonding and/or electrostatic interaction.
  • modifications include but are not limited to 2' position sugar modifications, locked nucleic acids, 5 position pyrimidine modifications, 8 position purine modifications, modification at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbone modifications, phophorothioate or alkyl phosphate modifications, methylations, unusual base-pairing combinations such as isobases isocytidine and isoguanidine, etc. Modifications can also include 3' and 5' modifications, such as capping. (See also Manoharan, Biochem. Biophys.
  • Oligonucleotides of the invention can be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
  • Enzymatic nucleic acids act by first binding to a target RNA (or DNA, see Cech U.S . Pat. No. 5,180,818). Such binding occurs through the target binding portion of an enzymatic nucleic acid which is held in close proximity to an enzymatic portion of molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein.
  • RNA aptamers There are at least five classes of ribozymes that each display a different type of specificity. For example, Group I Introns are -300 to >1000 nucleotides in size and require a U in the target sequence immediately 5' of the cleavage site and binds 4-6 nucleotides at the 5 '-side of the cleavage site. There are over 75 known members of this class, they are found in Tetrahymena thermophila rRNA, fungal mitochondria, chloroplasts, phage T4, blue-green algae, and others.
  • RNaseP RNA RNaseP RNA
  • Ml RNA RNaseP RNA
  • RNA portion of a ribonucleoprotein enzyme RNA portion of a ribonucleoprotein enzyme and cleave the tRNA precursors to form mature tRNA.
  • a third example are Hammerhead Ribozyme, which are -30 to 40 nucleotides in size. They require the target sequence UH immediately 5' of the cleavage site and bind a variable number nucleotides on both sides of the cleavage site.
  • RNA pathogens virusoids
  • a fourth class are the Hairpin Ribozymes, which are -50 nucleotides in size. They requires the target sequence GUC immediately 3' of the cleavage site and bind 4 nucleotides at the 5'-side of the cleavage site and a variable number to the 3'-side of the cleavage site. There are found in one plant pathogen (satellite RNA of the tobacco ringspot virus) which uses RNA as the infectious agent.
  • the fifth group are Hepatitis Delta Virus (HDV) Ribozymes, which are -60 nucleotides in size.
  • HDV Hepatitis Delta Virus
  • ribozyme can be advantageous over other technologies, such as antisense technology, in certain applications since the effective concentration of ribozyme necessary to effect a therapeutic treatment can be less than that of an antisense oligonucleotide.
  • This advantage reflects the ability of the ribozyme to act enzymatically.
  • a single ribozyme molecule is able to cleave many molecules of target RNA aptamers.
  • the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds.
  • the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA.
  • This cleavage mechanism is dependent upon factors additional to those involved in base pairing.
  • the specificity of action of a ribozyme under certain circumstances, can be greater than that of antisense oligonucleotide binding the same RNA site.
  • DNAzymes are single- stranded, and cleave both RNA and DNA.
  • a general model for the DNAzyme has been proposed, and is known as the "10-23" model.
  • DNAzymes following the "10-23” model, also referred to simply as “10-23 DNAzymes” have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each. In vitro analyses show that this type of DNAzyme can effectively cleave its substrate RNA at purine:pyrimidine junctions under physiological conditions.
  • DNAzyme means a DNA molecule that specifically recognizes and cleaves a distinct target nucleic acid sequence, which may be either DNA or RNA.
  • Ribozymes and DNAzymes comprise both substrate binding domains and catalytic domains. Rules for the optimal design of the regulatory ribozymes can be found within Methods in Molecular Biology Volume 74 "Ribozyme Protocols" edited by Philip C. Turner (Humana Press, Totowa, NJ, 1997). Strategies to identify reactive sites for ribozymes within a targeted nucleic acid are also described in this volume. Using these standard rules, hammerhead ribozymes, hairpin ribozymes, hepatitis delta virus ribozymes and ribozymes derived from group I introns can be specifically engineered to bind to and cleave a target nucleic acid ligand.
  • the ability of such ribozymes or DNAzymes to cleave the target nucleic acid ligand can be determined in any number of assays. For example, following incubation of a ribozyme or DNAzyme with an unlabeled or a labeled (eg. 32 P) target nucleic acid ligand under conditions favoring reaction, the target nucleic acid can be analyzed by denaturing gel electrophoresis to determine if the ribozyme or DNAzyme cleaved the backbone of the target nucleic acid ligand. Alternatively, fluorescence resonance energy transfer, or change in fluorescence intensity can be used to measure cleavage of an appropriately labeled nucleic acid Hgand.
  • PNAs Polynucleic Acid Analogues
  • MNAs Morpholino Nucleic Acids
  • LNAs Locked Nucleic Acids
  • PCOs Pseudo-Cyclic Oligonucleotides
  • Nucleobases of the oligonucleotide modulators of the invention can be connected via internucleobase linkages, e.g., peptidyl linkages (as in the case of peptide nucleic acids (PNAs); Nielsen et al. (1991) Science 254, 1497 and U.S. Pat. No. 5,539,082) and morpholino linkages (Qin et al., Antisense Nucleic Acid Drug Dev. 10, 11 (2000); Summerton, Antisense Nucleic Acid Drug Dev. 7, 187 (1997); Summerton et al., Antisense Nucleic Acid Drug Dev. 7, 63 (1997); Taylor et al., J Biol Chem.
  • internucleobase linkages e.g., peptidyl linkages (as in the case of peptide nucleic acids (PNAs); Nielsen et al. (1991) Science 254, 1497 and U.S. Pat. No. 5,539,082) and
  • the oHgonucleobases can also be Locked Nucleic Acids (LNAs). Nielsen et al., J Biomol Struct Dyn 17, 175 (1999); Petersen et al., J Mol Recognit 13, 44 (2000); Nielsen et al., Bioconjug Chem 11, 228 (2000).
  • LNAs Locked Nucleic Acids
  • PNAs are compounds that are analogous to oligonucleotides, but differ in composition. In PNAs, the deoxyribose backbone of oligonucleotide is replaced with a peptide backbone.
  • Each subunit of the peptide backbone is attached to a naturally-occurring or non-naturally-occurring nucleobase.
  • PNA often has an achiral polyamide backbone consisting of N-(2- aminoethyl)glycine units.
  • the purine or pyrimidine bases are linked to each unit via a methylene carbonyl linker (1-3) to target the complementary nucleic acid.
  • PNA binds to complementary RNA or DNA in a parallel or antiparallel orientation following the Watson-Crick base-pairing rules.
  • the uncharged nature of the PNA oligomers enhances the stability of the hybrid PNA/DNA(RNA) duplexes as compared to the natural homoduplexes.
  • PNA oligomers highly resistant to protease and nuclease attacks. These properties of PNA oligomers allow them to serve as efficient antisense or modulators of nucleic acid ligand activity. Indeed, peptide nucleic acids have been applied to block protein expression on the transcriptional and translational level, and microinjected PNA oligomers demonstrate a strong antisense effect in intact cells. See www.bioscience.org/1999/v4/d/soomets/fulltext.htm; and Frontiers in Bioscience, 4, d782-786 (Nov 1, 1999) for details on recent achievements on PNA antisense application, especially these concerned with whole cell or tissue delivery of the PNA.
  • PNA Peptide nucleic acids
  • PNAs bind to both DNA and RNA and form PNA/DNA or PNA RNA duplexes.
  • the resulting PNA/DNA or PNA/RNA duplexes are bound tighter than corresponding DNA/DNA or DNA/RNA duplexes as evidenced by their higher melting temperatures (T m ).
  • T m melting temperatures
  • This high thermal stability of PNA DNA(RNA) duplexes has been attributed to the neutrality of the PNA backbone, which results elimination of charge repulsion that is present in DNA/DNA or RNA/RNA duplexes.
  • Another advantage of PNA DNA(R A) duplexes is that T m is practically independent of salt concentration.
  • DNA/DNA duplexes are highly dependent on the ionic strength.
  • PNAs are an analogue of DNA in which the backbone is a pseudopeptide rather than a sugar, they mimic the behavior of DNA and bind complementary nucleic acid strands.
  • Unnatural nucleobases such as pseudo isocytosine, 5-methyl cytosine and 2,6-diaminopurine, among many others, also can be incorporated in PNA synthons.
  • PNAs are most commonly synthesized from monomers (PNA synthons) protected according to the t-Boc/benzyl protection strategy, wherein the backbone amino group of the growing polymer is protected with the t- butyloxycarbonyl (t-Boc) group and the exocyclic amino groups of the nucleobases, if present, are protected with the benzyloxycarbonyl (benzyl) group.
  • PNA synthons protected using the t- Boc/benzyl strategy are now commercially available.
  • PNA peptide nucleic acids
  • PNA PNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen bonding rules
  • Nature 1993, 365, 556-568 Buchardt, O., Egholm, M., Berg, R. & Nielsen, P.E. "Peptide Nucleic Acids (PNA) and their potential applications in medicine and biotechnology” Trends Biotechnology, 1993, 11, 384-386).
  • PNAs appear to be the very useful for a number of special applications.
  • PNAs can block translation anywhere in a mRNA by forming a double-clamp structure. With such rare RNA sequences one segment of the PNA binds to the target sequence by Watson/Crick bonds and the other segment of the PNA binds to major-groove sites of the resulting PNARNA duplex.
  • PNAs also readily form triple helix structures with rare duplex DNA sequences comprising mostly purines in one strand and mostly pyrimidines in the other strand.
  • PNAs under low salt conditions in cell-free systems PNAs have been shown to achieve sequence- specific invasion of duplex DNA sequences, resulting in inhibition of transcription of the invaded duplex.
  • Chinnery, P. F. et al. attached the presequence peptide of the nuclear-encoded human cytochrome c oxidase (COX) subunit VL ⁇ to biotinylated PNA which was successfully imported into isolated mitochondria in vitro (Chinnery, P. F. et al. 1999 Gene Ther. 6:1919-28). Delivery of the biotinylated peptide- PNA to mitochondria in intact cells was confirmed by confocal microscopy.
  • COX cytochrome c oxidase
  • a short hydrophobic peptide with the sequence biotinyl-FLFL coupled to a PNA trimer has been shown to internalize into human erythrocytes and Namalwa cells (Scarfi, S., A. Gasparini, G. Damonte & U. Benatti: Synthesis, uptake, and intracellular metabolism of a hydrophobic tetrapeptide- peptide nucleic acid (PNA)-biotin molecule. Biochem Biophys Res Commun 1997, 236, 323- 326). Basu and Wickstr ⁇ m (Synthesis and characterization of a peptide nucleic acid conjugated to aD-peptide analog of insulin-like growth factor 1 for increased cehular uptake.
  • Bioconjugate Chem 1997, 8, 481-488 showed that PNA conjugated to an all-D-amino acid insulin-like growth factor 1 (IGF1) mimicking peptide was specifically taken up by cells expressing the IGF1 receptor, although no antisense activity was described.
  • IGF1 insulin-like growth factor 1
  • US Patent No. 6,046,307 to Shay et al. discloses PNAs that inhibit telomerase activity in mammalian cells.
  • U.S. Patent No. 5,789,573 to Baker et al. disclose compositions and methods for inhibiting the translation of capped targeted mRNA using PNAs.
  • U.S. Patent No. 6, 165,720 disclose labeling of the PNA complex.
  • Morpholino nucleic acids are so named because they are assembled from morpholino subunits, each of which contains one of the four genetic bases (adenine, cytosine, guanine, and . thymine) linked to a 6-membered morpholine ring. Eighteen to twenty-five subunits of these four subunit types are joined in a specific order by non-ionic phosphorodiamidate intersubunit linkages to give a morpholino oligo.
  • Morpholinos devised by Summerton in 1985, constitute a radical redesign of natural nucleic acids, with the potential advantages of low cost starting materials and inexpensive assembly.
  • morpholinos are completely resistant to nucleases and they appear to be free of most or all of the non-antisense effects that are seen with S-DNAs. In contrast to PNAs, most morpholinos exhibit excellent aqueous solubility. Morpholinos also have much higher RNA binding affinities than do S-DNAs, though not as high as PNAs. Probably as a result of their substantial RNA binding affinities, long morpholinos (25-mers) provide predictable targeting and very high efficacy. Most notable, morpholinos provide good sequence specificity. The same factors that underlie their exceptional sequence specificity also render them unsuitable for targeting point mutations.
  • U.S. Patent No. 6,153,737 to Manoharan et al. is directed to derivatized oligonucleotides wherein the linked nucleosides are functionalized with peptides, proteins, water soluble vitamins or lipid soluble vitamins.
  • This disclosure was directed towards antisense therapeutics by modification of oligonucleotides with a peptide or protein sequence that aids in the selective entry of the complex into the nuclear envelope.
  • water-soluble and lipid-soluble vitamins can be used to assist in the transfer of the anti-sense therapeutic or diagnostic agent across cellular membranes.
  • PNAs and MNAs extremely attractive leads for the development of modulators of the present invention.
  • Peptide nucleic acids which can be conjugated to the carriers are distinguished in U.S. Patent No. 5,864,010 entitled Peptide Nucleic Acid Combinatorial Libraries and Improved Methods of Synthesis, developed by ISIS Pharmaceuticals, which is hereby incorporated by reference.
  • the peptide nucleic acids which can be conjugated to the carriers of the present invention are distinguished in U.S. Patent No. 5,986,053 entitled peptide nucleic acids complexes of two peptide nucleic acid strands and one nucleic acid strand.
  • LNA is a novel class of DNA analogues that possess some features that make it a prime candidate for modulators of the invention.
  • the LNA monomers are bi-cyclic compounds structurally similar to RNA-monomers. LNA share most of the chemical properties of DNA and RNA, it is water-soluble, can be separated by gel electrophoreses, ethanol precipitated etc (Tetrahedron, 54, 3607-3630 (1998)).
  • introduction of LNA monomers into either DNA or RNA oligos results in high thermal stability of duplexes with complementary DNA or RNA, while, at the same time obeying the Watson-Crick base-pairing rules.
  • PCOs Pseudo-cyclic oHgonucleobases
  • PCOs contain two oligonucleotide segments attached through their 3'-3' or 5'-5' ends.
  • One of the segments (the "functional segment") of the PCO has some functionality (e.g., an antisense oligonucleotide complementary to a target mRNA).
  • Another segment (the "protective segment") is complementary to the 3'- or 5'-terminal end of the functional segment (depending on the end through which it is attached to the functional segment).
  • PCOs form intramolecular pseudo-cyclic structures in the absence of the target nucleic acids (e.g., RNA).
  • PCOs are more stable than conventional antisense oligonucleotides because of the presence of 3 -3' or 5 -5' linkages and the formation of intramolecular pseudo-cyclic structures.
  • Pharmacokinetic, tissue distribution, and stability studies in mice suggest that PCOs have higher in vivo stability than and, pharmacokinetic and tissue distribution profiles similar to, those of PS-oligonucleotides in general, but rapid elimination from selected tissues.
  • a fluorophore and quencher molecules are appropriately linked to the PCOs of the present invention, the molecule will fluoresce when it is in the linear configuration, but the fluorescence is quenched in the cyclic conformation.
  • Protein-nucleic acid interactions are involved in many cellular functions, including transcription, RNA splicing, and translation. Synthetic molecules that can bind with high affinity to specific sequences of single-stranded nucleic acids have the potential to interfere with these interactions in a controllable way. Many proteins have been identified that can bind directly to nucleic acid sequences. One main group of proteins that directly bind to DNA are transcription factors.
  • transcription factors fall into the following classes: Basic domain transcription factors (leucine- zipper factors, CREB, helix-loop-helix factors, helix-loop-helix/ leucine zipper factors, cell-cycle controlling factors, NF-1, RF-X, bHSH), Zinc-coordinating DNA binding domain transcription factors (cys4 zinc finger of nuclear receptor type, thyroid hormone receptor-like factors, diverse Cys4 zinc fingers, Cys2His2 zinc finger domain, metabolic regulators in fungi, large factors with NF-6B-Hke binding properties, cys6 cysteine-zinc cluster, zinc fingers of alternating composition, homeo domain), Helix-turn-helix (homeo domain only, POU domain factors, paired box, fork head/ winged helix, heat shock factors, tryptophan clusters, TEA domain), beta- Scaffold Factors with minor groove contacts (RHR, STAT, p53, MADS box, beta-Barrel alpha- helix transcription factors, TATA
  • RNA In addition to its primary structure, RNA has the ability to fold into complex tertiary structures consisting of such local motifs as loops, bulges, pseudoknots, and turns. It is not surprising that, when they occur in RNAs that interact with proteins, these local structures are found to play important roles in protein-RNA interactions. This diversity of local and tertiary structure, however, makes it impossible to design synthetic agents with general, simple-to-use recognition rules analogous to those for the formation of double- and triple-helical nucleic acids. Tripeptides have been identified that are able to specifically bind to tertiary structures of nucleic acids (Hwang et al., Proc. Natl. Acad. Sci. USA 96:12997 (1999)). Thus, in one embodiment, these tripeptides may be used as modulators of nucleic acid ligand activity.
  • Peptide-based modulators of nucleic acid Hgands represent an alternative molecular class of modulators to oligonucleotides or their analogues. This class of modulators are particularly prove useful when sufficiently active oligonucleotide modulators of a target nucleic acid ligand can not be isolated due to the lack of sufficient single-stranded regions to promote nucleation between the target and the oligonucleotide modulator.
  • peptide modulators provide different bioavailabilities and pharmacokinetics than oligonucleotide modulators, and therefore, for some target nucleic acid ligands may be preferable.
  • Dye to the target nucleic acid ligand and incubating together the labeled-target and bead-immobilized peptide library under conditions in which binding between some members of the library and the nucleic acid are favored.
  • the binding of the nucleic acid ligand to the specific peptide on a given bead causes the bead to be "colored" by the label on the nucleic acid ligand, and thus enables the identification of peptides able to bind the target but simple isolation of the bead.
  • the direct interaction between peptides isolated by such screening methods and the target nucleic acid ligand can be confirmed and quantified using any number of the binding assays described to identify modulators of nucleic acid ligands.
  • the ability of said peptides to modulate the activity of the target nucleic acid ligand can be confirmed in appropriate bioassays.
  • the target nucleic acid ligand can be immobilized on a solid support (eg plastic, bead etc) by appending the appropriate affinity handle (eg biotin for streptavidin coated surfaces) or reactive site (eg. Aliphatic amine) to either end of the target nucleic acid ligand during chemical synthesis.
  • affinity handle eg biotin for streptavidin coated surfaces
  • reactive site eg. Aliphatic amine
  • biotinylated RNA or RNA containing the desired modification can be prepared according to standard transcription protocols, but including a 5-fold molar excess of a 5'-biotin-modified GMP or otherwise modified GMP over GTP in the reaction mixture.
  • peptides capable of binding said target RNA can be isolated by screening combinatorial peptide libraries, for example, random phage display libraries for peptides capable of binding the target nucleic acid ligand using standard methods for screening phage display libraries.
  • the direct interaction between peptides and the target nucleic acid ligand can be confirmed and quantified using any number of the binding assays described to identify potential modulators of nucleic acid ligands.
  • the ability of said peptides to modulate the activity of the target nucleic acid ligand can be confirmed in appropriate bioassays.
  • the mass spectral assays described can be used to screen amino acid and small molecular weight peptide libraries to identify those capable of binding to the target nucleic acid ligand (see U.S. Patent Nos. 6,342,393; 6,329,146; 6,253,168; and 6,221,587). Once identified, the direct interaction between such agents and the target nucleic acid ligand can be confirmed and quantified using any number of the binding assays described to identify potential modulators of nucleic acid ligands. The ability of said peptides to modulate the activity of the target nucleic acid ligand can be confirmed in appropriate bioassays.
  • USPs 5,834,184, 5,786,145 and 5,770,434 disclose methods for generating and screening for peptide modulators of the invention.
  • Oligosaccharides can interact with nucleic acids.
  • the antibiotic aminoglycosides are products of Streptomyces species and are represented by streptomycin, kanamycin, tobramycin, neomycin, netilmicin, amikacin and gentamicin. These antibiotics exert their activity by binding to bacterial ribosomes and preventing the initiation of protein synthesis.
  • Aminoglycoside antibiotics interact specifically with diverse RNA molecules such as various ribozymes and the HTV-l's TAR and RRE sequences. Aminoglycoside antibiotics that bind to the 30s ribosomal A- site RNA cause misreading of the genetic code and inhibit translocation.
  • the aminoglycoside antibiotic paromomycin binds specifically to the RNA oligonucleotide at the 30S A site. This antibiotic binds the major groove on the A-site RNA within a pocket created by an A-A pair and a single bulged adenine. There are several interactions that occur between the aminoglycoside chemical groups and the conserved nucleotides in the RNA.
  • oligosaccharides like aminoglycosides, can bind to nucleic acids and can be used to modulate the activity of nucleic acid ligands.
  • the mass spectral assays described can be used to screen for oligosaccharides (e.g., aminoglycosides), alone or in a mixture, that are capable of binding to the target nucleic acid ligand (see U.S. Patent Nos. 6,342,393; 6,329,146; 6,253,168; and 6,221,587).
  • a small molecule that intercalates between the nucleic acid Hgand and the target or otherwise disrupts or modifies the binding between the nucleic acid Hgand and target can also be used as the therapeutic regulator.
  • Such small molecules can be identified by screening candidates in an assay that measures binding changes between the nucleic acid ligand and the target with and without the small molecule, or by using an in vivo or in vitro assay that measures the difference in biological effect of the nucleic acid ligand for the target with and without the small molecule. Once a small molecule is identified that exhibits the desired effect, techniques such as combinatorial approaches can be used to optimize the chemical structure for the desired regulatory effect.
  • oUgonucleotides or analogues thereof including ribozymes or DNAzymes or peptide nucleic acids (PNAs) or mopholino nucleic acids (MNAs)
  • nucleic acid binding peptides, polypeptides or proteins including nucleic acid binding tripeptides, oligosaccharides, and small molecules
  • PNAs ribozymes or DNAzymes or peptide nucleic acids
  • MNAs mopholino nucleic acids
  • nucleic acid binding peptides including nucleic acid binding tripeptides, oligosaccharides, and small molecules
  • an oligonucleotide, PNA or MNA can be linked to a peptide or protein to provide desired properties or therapeutic effect.
  • an oligonucleotide or analogue thereof or peptide or protein can be linked to a small molecule to enhance its properties.
  • the small molecule includes a radioligand or other detectable agent that can be monitored using standard techniques. In this way, the progress and location of the regulator can be monitored.
  • the small molecule is a therapeutic that is brought to the site of therapy by the regulator, and then optionally cleaved to provide its therapeutic effect at the appropriate location.
  • the small molecule is a chelator that joins two biological materials together to produce a combined effect. The chimeras, fusion products or otherwise multicomponent regulator can be identified and evaluated for therapeutic efficacy as described in detail below.
  • Standard binding assays can be used to identify and select modulators of the invention.
  • Nonlimiting examples are gel shift assays and BIACORE assays. That is, test modulators can be contacted with the nucleic acid ligands to be targeted under test conditions or typical physiological conditions and a determination made as to whether the test modulator in fact binds the nucleic acid Hgand. Test modulators that are found to bind the nucleic acid ligand can then be analyzed in an appropriate bioassay (which will vary depending on the aptamer and its target molecule, for example coagulation tests) to determine if the test modulator can affect the biological effect caused by the nucleic acid ligand on its target molecule.
  • an appropriate bioassay which will vary depending on the aptamer and its target molecule, for example coagulation tests
  • the Gel-Shift assay is a technique used to assess binding capability. For example, a DNA fragment containing the test sequence is first incubated with the test protein or a mixture containing putative binding proteins, and then separated on a gel by electrophoresis. If the DNA fragment is bound by protein, it will be larger in size and its migration will therefore be retarded relative to that of the free fragment.
  • one method for a electrophoretic gel mobility shift assay can be (a) contacting in a mixture a nucleic acid binding protein with a non- radioactive or radioactive labeled nucleic acid molecule comprising a molecular probe under suitable conditions to promote specific binding interactions between the protein and the probe in forming a complex, wherein said probe is selected from the group consisting of dsDNA, ssDNA, and RNA; (b) electrophoresing the mixture; (c) transferring, using positive pressure blot transfer or capillary transfer, the complex to a membrane, wherein the membrane is positively charged nylon; and (d) detecting the complex bound to the membrane by detecting the non-radioactive or radioactive label in the complex.
  • the Biacore technology measures binding events on the sensor chip surface, so that the interactant attached to the surface determines the specificity of the analysis. Testing the specificity of an interaction involves simply analyzing whether different molecules can bind to the immobilized interactant. Binding gives an immediate change in the surface plasmon resonance (SPR) signal, so that it is directly apparent whether an interaction takes place or not.
  • SPR-based biosensors monitor interactions by measuring the mass concentration of biomoiecules close to a surface. The surface is made specific by attaching one of the interacting partners. Sample containing the other partner(s) flows over the surface: when molecules from the sample bind to the interactant attached to the surface, the local concentration changes and an SPR response is measured. The response is directly proportional to the mass of molecules that bind to the surface.
  • SPR arises when light is reflected under certain conditions from a conducting film at the interface between two media of different refractive index.
  • the media are the sample and the glass of the sensor chip, and the conducting film is a thin layer of gold on the chip surface.
  • SPR causes a reduction in the intensity of reflected light at a specific angle of reflection. This angle varies with the refractive index close to the surface on the side opposite from the reflected light.
  • concentration and therefore the refractive index at the surface changes and an SPR response is detected. Plotting the response against time during the course of an interaction provides a quantitative measure of the progress of the interaction.
  • the Biacore technology measures the angle of minimum reflected light intensity.
  • SPR response values are expressed in resonance units (RU).
  • RU resonance units
  • One RU represents a change of 0.0001° in the angle of the intensity minimum. For most proteins, this is roughly equivalent to a change in concentration of about 1 pg/mm2 on the sensor surface. The exact conversion factor between RU and surface concentration depends on properties of the sensor surface and the nature of the molecule responsible for the concentration change.
  • oligonucleotide or analogue thereof, peptide, polypeptide, oligosaccharide or small molecule can bind to the nucleic acid ligand in a manner such that the interaction with the target is modified.
  • assays for example, electrophoretic mobility shift assays (EMSAs), titration calorimetry, scintillation proximity assays, sedimentation equilibrium assays using analytical ultracentrifugation (see for eg.
  • fluorescence polarization assays fluorescence anisotropy assays, fluorescence intensity assays, fluorescence resonance energy transfer (FRET) assays, nitrocellulose filter binding assays, ELISAs, ELONAs (see, for example, USP 5,789,163), RIAs, or equilibrium dialysis assays can be used to evaluate the ability of an agent to bind to a nucleic acid ligand.
  • Direct assays in which the interaction between the agent and the nucleic acid ligand is directly determined can be performed, or competition or displacement assays in which the ability of the agent to displace the nucleic acid ligand from its target can be performed (for example, see Green, Bell and Janjic, Biotechniques 30(5), 2001, p 1094 and USP 6,306,598).
  • a candidate modulating agent Once a candidate modulating agent is identified, its ability to modulate the activity of a nucleic acid ligand for its target can be confirmed in a bioassay.
  • binding assays can be used to verify that the agent is interacting directly with the nucleic acid ligand and can measure the affinity of said interaction.
  • mass spectrometry can be used for the identification of an regulator that binds to a nucleic acid ligand, the site(s) of interaction between the regulator and the nucleic acid ligand, and the relative binding affinity of agents for the nucleic acid ligand (see for example U.S. Patent No. 6,329,146, Crooke et al).
  • Such mass spectral methods can also be used for screening chemical mixtures or libraries, especially combinatorial libraries, for individual compounds that bind to a selected target nucleic acid ligand that can be used in as modulators of the nucleic acid ligand.
  • mass spectral techniques can be used to screen multiple target nucleic acid ligands simultaneously against, e.g. a combinatorial library of compounds.
  • mass spectral techniques can be used to identify interaction between a plurality of molecular species, especially "small" molecules and a molecular interaction site on a target nucleic acid ligand.
  • coagulation tests can be performed as bioassays in clinical laboratories. These tests are generally functional end-point assays, in which a patient sample (plasma or whole blood) is incubated with exogenous reagents that activate the coagulation cascade, and the time until clot formation is measured. The clotting time of the patient sample is then compared to the clotting time of pooled normal plasma or whole blood to provide a standard measurement of the patient's hemostatic status. As described below, such clotting assays are commonly used as screening tests that evaluate the functioning of both the patient's intrinsic and extrinsic coagulation systems.
  • ACT Activated Clotting Time Test
  • APTT activated partial thromboplastin time
  • ACT can be used to monitor a patient's coagulation status in connection with clinical procedures, such as those that involve the administration of high doses of heparin (e.g., CPB and PTCA).
  • the Activated Partial Thromboplastin Time Test is a common central laboratory test, APTT is used to evaluate the intrinsic coagulation pathway, which includes factors I, U, V, Vi ⁇ , LX, X, XI, and 2 II.
  • the test is performed using a plasma sample, in which the intrinsic pathway is activated by the addition of phospholipid, an activator (ellagic acid, kaolin, or micronized silica), and Ca 2+ . Formation of the Xase and prothrombinase complexes on the surface of the phospholipid enables protbrombin to be converted into thrombin, with subsequent clot formation.
  • the result of the APTT test is the time (in seconds) required for this reaction.
  • APTT can be used to assess the overall competence of a patient's coagulation system, as a preoperative screening test for bleeding tendencies, and as a routine test for monitoring heparin therapy.
  • Another example of the APTT is performed as follows. First, blood plasma is collected after the blood was subjected to centrifugal separation, and then acting is added thereto. In addition, calcium chloride is added. The period of time is measured until coagulation is formed More in detail, the blood plasma is stored in a refrigerator after it was obtained through centrifugal separation of blood. Activating agent of 0.1 ml, which was warmed in a water having a temperature of 37° C.
  • the bleeding time test can be used for the diagnosis of hemostatic dysfunction, von Willebrand's disease, and vascular disorders. It also can be used to screen for platelet abnormalities prior to surgery. The test is performed by making a small incision on the forearm and wicking away the blood from the wound site. The time it takes for bleeding to stop is recorded and in control subjects is approximately 3.5 minutes. Prolongation of the bleeding time is indicative of qualitative or quantitative platelet defects.
  • the Prothrombin Time Test (PT), which was first described by Quick in 1935, measures the tissue factor-induced coagulation time of blood or plasma. It is used as a screening test to evaluate the integrity of the extrinsic coagulation pathway, and is sensitive to coagulation factors I, Tl, V, VII, and X.
  • the test can be performed by adding thromboplastin and Ca 2+ to a patient sample and measuring the time for clot formation.
  • a prolonged clotting time suggests the presence of an inhibitor to, or a deficiency in, one or more of the coagulation factors of the extrinsic pathway.
  • PT clotting time can also be prolonged for patients on warfarin therapy, or for those with vitamin K deficiency or liver dysfunction.
  • the PT test can provide an assessment of the extrinsic coagulation pathway, and is widely used to monitor oral anticoagulation therapy.
  • the Thrombin Clotting Time Test measures the rate of a patient's clot formation compared to that of a normal plasma control.
  • the test can be performed by adding a standard amount of thrombin to a patient's plasma that has been depleted of platelets, and measuring the time required for a clot to form. This test has been used as an aid in the diagnosis of disseminated intravascular coagulation (DIC) and liver disease.
  • DIC disseminated intravascular coagulation
  • Complex Tests include specific factor assays based on laboratory tests, such as the APTT, PT, and TCT tests.
  • One assay measures the level of the activation peptide factor TXa or the factor IXa-antithrombin III complex. These measurements are used to determine the levels of factor IXa or factor VTI-tissue mediated complex.
  • Assays for activated protein C resistance, antithrombin, protein C deficiency, and protein S deficiency are also part of this group. Asymptomatic individuals who have heterogeneous deficiencies of proteins C and S, and resistance to activated protein C, have significantly elevated levels of the prothrombin fragment F1.2 compared to controls.
  • a method of modulating biological activity includes (i) administering to a patient in need thereof a nucleic acid ligand that binds to a target to produce a therapeutic effect, and then at the selected or desired time, (ii) administering to the patient a modulator that modifies the therapeutic effect.
  • the modulator turns off the therapeutic effect.
  • the modulator reduces or minimizes but does not terminate the therapeutic effect.
  • the modulator enhances the therapeutic effect.
  • the base therapeutic effect is determined by the target and the nucleic acid Hgand.
  • the modification of the therapeutic effect is determined by the modulator. Any known or developed nucleic acid ligand can be regulated in accordance with this invention.
  • the method comprises: (a) administering to a patient, including any warm-blooded vertebrate in need thereof, an effective amount of a nucleic acid ligand or DNA aptamer that selectively binds a coagulation pathway factor, the RNA aptamer having a dissociation constant for the coagulation pathway factor of about 20 nM or less; (b) modulating the biological activity of the coagulation pathway factor in the warm-blooded vertebrate through the administering of the RNA aptamer in step (a); and (c) providing an antidote to reverse the effects of the aptamer by administration of a modulator.
  • the modulators of the present invention can bind to nucleic acid Hgands that target tissue factor (TF)/factor Vila (FVHa), factor VIHa (FVHIaVfactor LXa (FLXa), factor Va (FVa/factor Xa (Fxa) enzyme complexes and platelet receptors such as gp ⁇ bJJIa and gp IbLX and modulate the effects of the nucleic acid ligand.
  • This invention also provides antidote control of platelet inhibitors, antithrombotics and fibrinolytics.
  • the first case is when anticoagulant or antithrombotic treatment leads to hemorrhage, including intracranial or gastrointestinal hemorrhage. While identifying safer target proteins may reduce this risk, the potential for morbidity or mortality from this type of bleeding event is such that the risk can not be overlooked.
  • the second case is when emergency surgery is required for patients who have received antithrombotic treatment. This clinical situation arises in a low percentage of patients who require emergency coronary artery bypass grafts while undergoing percutaneous coronary intervention under the coverage of GPIIb/Tfla inhibitors.
  • the third case is when an anticoagulant nucleic acid ligand is used during a cardiopulmonary bypass procedure. Bypass patients are predisposed to post operative bleeding.
  • an antidote e.g., an oligonucleotide modulator of the invention targeted to an anticoagulant or antithrombotic nucleic acid ligand
  • an antidote e.g., an oligonucleotide modulator of the invention targeted to an anticoagulant or antithrombotic nucleic acid ligand
  • a method of treating cardiovascular disease in patients comprises administering an effective amount of an RNA aptamer that selectively binds a coagulation pathway factor, the RNA aptamer having a dissociation constant for the coagulation pathway factor of about 20 nM or less, to a vertebrate subject suffering from cardiovascular disease, whereby cardiovascular disease in the vertebrate subject is treated, then providing an antidote to reverse the effects of the aptamer by administration of a modulator.
  • a method of modulating E2F activity in patient, including a warm-blooded vertebrate, in which such modulation is desired comprises: (a) administering to the warm-blooded vertebrate an effective amount of an RNA aptamer that selectively binds an E2F family member, the RNA aptamer having a dissociation constant for the E2F family member of about 20 nM or less; (b) modulating E2F in the warm-blooded vertebrate through the administering of the RNA aptamer of step (a); and (c) providing an antidote to reverse the effects of the aptamer by administration of a modulator.
  • the patient treated in the present invention in its many embodiments is desirably a human patient, although it is to be understood that the principles of the invention indicate that the invention is effective with respect to all vertebrate species, including warm-blood vertebrates (e.g., birds and mammals), which are intended to be included in the term "patient".
  • a mammal is understood to include any mammalian species in which treatment of cardiovascular disease is desirable, particularly agricultural and domestic mammalian species.
  • Contemplated is the treatment of mammals such as humans, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economical importance (animals raised on farms for consumption by humans and/or social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans such as cats and dogs), swine (pigs, hogs, and wild board), ruminants (such as cattle, oxen, sheep, giraffes, deer goats, bison, and camels), and horses.
  • fowl i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economical importance to humans.
  • the present method for treating cardiovascular disease in a tissue contemplates contacting a tissue in which cardiovascular disease is occurring, or is at risk for occurring, with a composition comprising a therapeutically effective amount of an RNA aptamer capable of binding a coagulation factor as well as providing an antidote to reverse the effects of the aptamer by administration of a modulator.
  • the method comprises administering to a patient a therapeutically effective amount of a physiologically tolerable composition containing the RNA aptamer as well as a method to provide an antidote to reverse the effects of the aptamer by administration of a modulator.
  • the dosage ranges for the administration of the modulator depend upon the form of the modulator, and its potency, as described further herein, and are amounts large enough to produce the desired effect.
  • the dosage should not be so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The individual physician in the event of any complication can also adjust the dosage.
  • a therapeutically effective amount is an amount of a modulator sufficient to produce a measurable modulation of the effects of the nucleic acid ligand, including but not limited to a coagulation-modulating amount, an E2F activity-modulating amount coagulation, and/or angiogenesis factor activity (e.g., Angl or Ang2 activity)-modulating amount.
  • Modulation of coagulation, E2F activity, and/or angiogenesis .factor activity can be measured in situ by immunohistochemistry by methods disclosed in the Examples, or by other methods known to one skilled in the art.
  • a preferred modulator has the ability to substantially bind to a nucleic acid ligand in solution at modulator concentrations of less than one (1.) micromolar ( ⁇ M), preferably less than 0.1 ⁇ M, and more preferably less than 0.01 ⁇ M.
  • ⁇ M micromolar
  • substantially is meant that at least a 50 percent reduction in target biological activity is observed by modulation in the presence of the a target, and at 50% reduction is referred to herein as an IC50 value.
  • Preferred modes of administration of the materials of the present invention to a mammalian host are parenteral, intravenous, intradermal, intra-articular, intra-synovial, intrathecal, intra-arterial, intracardiac, intramuscular, subcutaneous, intraorbital, intracapsular, intraspinal, intrasternal, topical, transdermal patch, via rectal, vaginal or urethra! suppository, peritoneal, percutaneous, nasal spray, surgical implant, internal surgical paint, infusion pump or via catheter.
  • the agent and carrier are administered in a slow release formulation such as an implant, bolus, microparticle, microsphere, nanoparticle or nanosphere.
  • modulators of the present invention can be preferably administered parenterally by injection or by gradual infusion over time.
  • tissue to be treated can typically be accessed in the body by systemic administration and therefore most often treated by intravenous administration of therapeutic compositions, other tissues and delivery techniques are provided where there is a likelihood that the tissue targeted contains the target molecule.
  • modulators of the present invention are typically administered orally, topically to a vascular tissue, intravenously, intraperitoneally, intramuscularly, subcutaneously, intra-cavity, transdermally, and can be delivered by peristaltic techniques.
  • compositions can be provided to the individual by a variety of routes such orally, topically to a vascular tissue, intravenously, intraperitoneally, intramuscularly, subcutaneously, intra-cavity, transdermally, and can be delivered by peristaltic techniques.
  • non- liming approaches for topical administration to a vascular tissue include (1) coating or impregnating a blood vessel tissue with a gel comprising a nucleic acid Hgand, for delivery in vivo, e.g., by implanting the coated or impregnated vessel in place of a damaged or diseased vessel tissue segment that was removed or by-passed; (2) delivery via a catheter to a vessel in which delivery is desired; (3) pumping a nucleic acid ligand composition into a vessel that is to be implanted into a patient.
  • the nucleic acid ligand can be introduced into cells by microinjection, or by Hposome encapsulation.
  • nucleic acid ligands of the present invention can be administered in a single daily dose, or the total daily dosage can be administered in several divided doses. Thereafter, the modulator is provided by any suitable means to alter the effect of the nucleic acid ligand by administration of the modulator.
  • the therapeutic compositions comprising modulator polypeptides of the present invention are conventionally administered intravenously, as by injection of a unit dose, for example.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier or vehicle.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • quantity to be administered depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual.
  • suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for administration are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.
  • compositions, carriers, diluents and reagents are used interchangeably and represent that the materials are capable of administration without substantial or debilitating toxic side effects.
  • compositions comprising a modulator of the present invention can be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation can be found in Remingto 's Pharmaceutical Sciences.
  • a pharmaceutically acceptable composition suitable for effective administration such compositions will contain an effective amount of the aptamer.
  • Such compositions can contain admixtures of more than one modulator.
  • the effective amount can vary according to a variety of factors such as the individual's condition, weight, sex, age and amount of nucleic acid lidgand administered. Other factors include the mode of administration.
  • the compositions will be administered in dosages adjusted for body weight, e.g., dosages ranging from about 1 ⁇ g/kg body weight to about 100 mg/kg body weight, preferably, 1 g/kg body weight to 50 mg kg body weight.
  • Modulators of nucleic acid ligands can be particularly useful for the treatment of diseases where it is beneficial to inhibit coagulation, E2F activity, and/or angiogenesis factor activity (e.g., Angl or Ang2 activity), or prevent such activity from occurring.
  • angiogenesis factor activity e.g., Angl or Ang2 activity
  • the pharmaceutical compositions are administered in therapeutically effective amounts, that is, in amounts sufficient to generate a coagulation-, E2F activity-, and/or angiogenesis factor activity (e.g., Angl or Ang2 activity )-modulating response, or in prophylactically effective amounts, that is in amounts sufficient to prevent a coagulation factor from acting in a coagulation cascade, to prevent an E2F activity-mediated response, or to prevent an angiogenesis factor activity (e.g., Angl or Ang2 activity )-mediated response.
  • the therapeutically effective amount and prophylactically effective amount can vary according to the type of modulator.
  • the pharmaceutical composition can be administered in single or multiple doses.
  • oligonucleotide modulators of the invention can be administered using established protocols used in antisense therapies.
  • the data presented in Example 6 indicate the activity of a therapeutic nucleic acid ligand can be modulated by the intravenous infusion of antidote oligonucleotides into a human or other animal.
  • the modulator's activity is durable, once the desired level of modulation of the nucleic acid ligand by the modulator is achieved, infusion of the modulator can be terminated, allowing residual modulator to clear the human or animal. This allows for subsequent re-treatment with the nucleic acid ligand as needed.
  • subsequent treatment can involve the use of a second, different nucleic acid ligand/modulator (e.g., oligonucleotide) pair .
  • Modulators synthesized or identified according to the methods disclosed herein can be used alone at appropriate dosages defined by routine testing in order to obtain optimal modulation of nucleic acid ligand activity in coagulation, E2F, and/or angiogenesis factor cascades (e.g., Angl or Ang2 activity) while minimizing any potential toxicity.
  • angiogenesis factor cascades e.g., Angl or Ang2 activity
  • co- administration or sequential administration of other agents can be desirable.
  • the active agents can be administered concurrently, or they each can be administered at separately staggered times.
  • the dosage regimen utilizing the modulators of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular modulator employed.
  • a physician of ordinary skill can readily determine and prescribe the effective amount of the aptamer required to prevent, counter or arrest the progress of the condition.
  • Optimal precision in achieving concentrations of modulator within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the modulator's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of the modulator.
  • the modulators herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as "carrier” materials) suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrup, suppositories, gels and the like, and consistent with conventional pharmaceutical practices.
  • carrier suitable pharmaceutical diluents, excipients or carriers
  • the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture.
  • suitable binders include without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like.
  • Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.
  • suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like.
  • Other dispersing agents that can be employed include glycerin and the like.
  • parenteral administration sterile suspensions and solutions are desired. Isotonic preparations that generally contain suitable preservatives are employed when intravenous administration is desired.
  • Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 mydstyl propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.
  • carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 mydstyl propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.
  • the compounds of the present invention can also be administered in the form of Hposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
  • the compounds of the present invention can also be coupled with soluble polymers as targetable drug carriers.
  • soluble polymers can include polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-amidephenol, polyhydroxy-ethylaspartamidepbenol, or polyethyl- eneoxidepolylysine substituted with palmitoyl residues.
  • the compounds of the present invention can be coupled (preferably via a covalent linkage) to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polyethylene glycol (PEG), polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
  • PEG polyethylene glycol
  • polylactic acid polyepsilon caprolactone
  • polyhydroxy butyric acid polyorthoesters
  • polyacetals polydihydro-pyrans
  • polycyanoacrylates polycyanoacrylates
  • cross-linked or amphipathic block copolymers of hydrogels for example, polyethylene glycol (PEG), polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans
  • the oligonucleotide modulators can be administered directly (e.g., alone or in a liposomal formulation or complexed to a carrier (e.g., PEG)) (see for example, USP 6,147,204, USP 6,011,020).
  • a carrier e.g., PEG
  • the nucleic acid ligand or modulator can be comprised of a oligonucleotide modulator attached to a non-immunogenic, high molecular weight compound such as polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the pharmacokinetic properties of the complex are improved relative to the nucleic acid ligand alone.
  • the association could be through covalent bonds or non-covalent interactions.
  • the modulator is associated with the PEG molecule through covalent bonds.
  • PEG may be covalently bound to a variety of positions on the oligonucleotide modulator.
  • an oligonucleotide modulator is bonded to the 5' thiol through a maleimide or vinyl sulfone functionality.
  • a plurality of modulators can be associated with a single PEG molecule.
  • the modulator can be to the same or different target.
  • a plurality of PEG molecules can be attached to each other.
  • one or more modulators to the same target or different targets can be associated with each PEG molecule. This also results in an increase in avidity of each modulator to its target.
  • nucleic acids One problem encountered in the therapeutic and in vivo diagnostic use of nucleic acids is that oligonucleotides in their phosphodiester form may be quickly degraded in body fluids by intracellular and extracellular enzymes such as endonucleases and exonucleases before the desired effect is manifest. Certain chemical modifications of the nucleic acid can be made to increase the in vivo stability of the nucleic acid or to enhance or to mediate the delivery of the nucleic acid.
  • nucleic acid ligands contemplated in this invention include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid bases or to the nucleic acid as a whole.
  • modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, unusual base-pairing combinations such as the isobases isocytidine and isoguanidine and the like. Modifications can also include 3' and 5' modifications such as capping.
  • Lipophilic compounds and non-immunogenic high molecular weight compounds with which the modulators of the invention can be formulated for use in the present invention can be prepared by any of the various techniques presently known in the art or subsequently developed.
  • they are prepared from a phospholipid, for example, distearoyl phosphatidylcholine, and may include other materials such as neutral lipids, for example, cholesterol, and also surface modifiers such as positively charged (e.g., sterylamine or aminomannose or aminomannitol derivatives of cholesterol) or negatively charged (e.g., diacetyl phosphate, phosphatidyl glycerol) compounds.
  • Multilamellar liposomes can be formed by the conventional technique, that is, by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase is then added to the vessel with a swirling or vortexing motion which results in the formation of ML Vs.
  • UVs can then be formed by homogenization, sonication or extrusion (through filters) of MLV's. In addition, UVs can be formed by detergent removal techniques.
  • the complex comprises a Hposome with a targeting nucleic acid ligand(s) associated with the surface of the Hposome and an encapsulated therapeutic or diagnostic agent.
  • Preformed liposomes can be modified to associate with the nucleic acid ligands.
  • a cationic Hposome associates through electrostatic interactions with the nucleic acid.
  • a nucleic acid attached to a lipophilic compound, such as cholesterol can be added to preformed liposomes whereby the cholesterol becomes associated with the liposomal membrane.
  • the nucleic acid can be associated with the Hposome during the formulation of the Hposome.
  • the nucleic acid is associated with the Hposome by loading into preformed liposomes.
  • oligonucleotide modulators of the invention can be produced in vivo following administration of a construct comprising a sequence encoding the oligonucleotide.
  • Techniques available for effecting intracellular delivery of RNA modulators of gene expression can be used (see generally Sullenger et al, Mol. Cell Biol. 10:6512 (1990)).
  • RNA 9.3t (t" for truncate), shown in Fig. 1.
  • This 34 nucleotide aptamer has a molecular weight of 11.5 kDa and binds FLXa with essentially the same affinity as the full-length sequence (K D 0.6 nM).
  • K D 0.6 nM full-length sequence
  • a mutant version of RNA 9.3t termed 9.3tM was synthesized in which the absolutely conserved A's in the internal loop were mutated to G's (Fig. 1).
  • This aptamer binds FLXa with a K D > 5 ⁇ M as determined by competition binding assays.
  • RNA 9.3tM is employed as a control to measure any non-specific effects caused by an aptamer of this composition.
  • Aptamer 9.3t blocks FX activation by FVUIa FIXa/lipids, and also partially blocks synthetic substrate hydrolysis by FLXa.
  • the affinity of 9.3t for FIX, FVJJa, FXa, FXIa and APC was measured in direct binding assays as previously described (Rusconi et al, Thrombosis and Haemostasis 83:841-848 (2000)).
  • the aptamer binds FIX only -5-50 fold less tightly than FLXa. It failed to exhibit significant binding at protein concentrations up to 5 ⁇ M (fraction RNA bound ⁇ 10%) to any of the other proteins tested. Therefore, the specificity of aptamer 9.3t for FLXa versus FVTfa, FXa, FXIa or APC is >5000 fold.
  • RNA 9.3t is a potent anticoagulant, with a maximal effect on the APTT similar to that observed in FIX deficient plasma. Similar experiments have been performed in porcine plasma, and similar potency and specificity have been observed.
  • this aptamer to systemically anticoagulate small (1.5-4 kg) pigs following bolus intravenous injection was tested.
  • an intravenous catheter was placed in the femoral vein for sample injection and an arterial catheter was placed in a femoral artery of the animal for serial withdrawal of blood samples.
  • a pre-injection blood sample was taken, and the ACT time measured on site to establish a baseline whole blood clot time for the animal.
  • 9.3t-C a version of this aptamer with a 5 'cholesterol moiety was synthesized, termed 9.3t-C.
  • the cholesterol modifed aptamer retained high affinity binding to FIXa (Fig. 5A) and potent anticoagulant activity (Figs. 5B and 5C).
  • the ACT of the animal increased -1.4 fold and was sustained at this level for 1 hour post injection (Fig. 6A).
  • Fig. 6B shows analysis of the APTT and PT of the animals from this experiment. While the anticoagulant potency of the two aptamers is similar, the duration of the anticoagulant effect of 9.3t-C is significantly longer (Fig. 6C).
  • the secondary structure model of 9.3t is shown in Fig. 1 and was developed from comparative sequence analysis of the related FLXa aptamer sequences shown in Fig. 7. These data strongly support formation of the stem-looped structure depicted in Fig. 1.
  • mutational analysis of aptamer 9.3t demonstrated that disruption of either stem 1 or stem 2 resulted in a greater than 1000 fold loss of affinity for FLXa. Therefore, a 17 residue all 2'Omethyl oligonucleotide was designed (sequence 5' auggggaggcagcauua 3') (Anti-Dl) complementary to the 3 'half of aptamer 9.3t beginning at the 5' end of loop 2 (L2 in Fig.
  • radiolabeled aptamer 9.3t (125 nM) was incubated with increasing concentrations of the "antidote" oligonucleotide (from equimolar to an 8 fold molar excess) at 37°C for 15 minutes (-heat Fig 8), and the amount . of intermolecular duplex formed was visualized by native gel electrophoresis (12% acrylamide, 150 mM NaCI, 2 mM CaCl 2 , run in lXtris-borate buffer +2 mM CaCI 2 ) followed by phosphorimaging (Fig. 8).
  • oligonucleotide-aptamer complex was annealed to aptamer 9.3t by heating the aptamer and an 8 fold molar excess of the oligonucleotide at 95°C for 5 minutes prior to the 37°C incubation (+heat Fig. 8).
  • the same set of experiments was performed with a nonsense oligonucleotide of the same base composition as the antidote oligonucleotide (N.S. in Fig. 8).* As can be seen in Fig.
  • the antidote oligonucleotide readily denatures aptamer 9.3t as evidenced by near complete formation of the oligonucleotide-aptamer complex when the oligonucleotide was present at an 8 fold molar excess to the aptamer.
  • this interaction is very specific, as no complex is observed, with or without heating, between the aptamer and the nonsense control oHgonucleotide.
  • aptamer 9.3t was pre-incubated 5 minutes in plasma prior to addition of the antidote oligonucleotide to generate aptamer-FLX complexes, the antidote or nonsense oHgonucleotide were then added, and the incubation continued for an additional 10 minutes prior to adding CaCl 2 to initiate clot formation. As shown in Figs.
  • the antidote oligonucleotide is able to effectively reverse about 80% of the anticoagulant activity of aptamer 9.3t.
  • the molar excess of antidote oHgonucleotide required to achieve this effect is substantially larger than the amount required to effectively denature the aptamer in the absence of protein.
  • aptamer 9.20t was prepared (see Fig. 10A). Stem 1 of 9.20t is identical to stem 1 of aptamer 9.3t, as is the 3' half of stem 2. The main differences between 9.20t and 9.3t are found in loop 2 and loop 3 (compare Fig. 1 with Fig. 10A).
  • Aptamer 9.20t binds FIXa with a K ⁇ comparable to that of 9.3t.
  • APTT assays were used to measure the clot time of human plasma as a function of the concentration of aptamer 9.20t. Its in vitro anticoagulant potency in human plasma was comparable to that of 9.3t (Fig. 10B).
  • Aptamer 9.20t was added to human plasma at a concentration of 50 nM and allowed to bind to plasma FLX for 5 minutes. Varying concentrations of antidote oligonucleotide Anti DI were then added, and the APTT was measured at 10 minutes after antidote addition to plasma. The relative change in clot time caused by 9.20t addition to plasma was unaffected by the addition of this antidote oHgonucleotide complimentary to aptamer 9.3t (Fig. 10C).
  • a 3' tail was added to aptamer 9.3t, the tailed ' aptamer being designated 9.3t-3NT (Fig. 11).
  • the 3' tail of 9.3t-3NT is a 3 nucleotide 2'Omethyl modified RNA tail. This tail sequence was chosen to reduce the likelihood of impacting the activity of the aptamer, and to reduce potential secondary structures within the complimentary antidote oligonucleotide.
  • the affinity of aptamer 9.3t-3NT was compared to 9.3t in competition binding assays (Fig. 12A).
  • the affinity of aptamer 9.3t-3NT for FLXa was comparable to that of 9.3t (K D 1.5 nM or less).
  • APTT assays were used to measure the clot time of human plasma as a function of the concentration of aptamers 9.3t-3NT and 9.3t (Fig. 12B).
  • the anticoagulant activities of the aptamers were similar, and both aptamers were able to completely inhibit FIX activity in human plasma.
  • Aptamer 9.3t-3NT was added to human plasma at a concentration of 50 nM (-3 fold increase in APTT) and allowed to bind to plasma FLX for 5 minutes. Varying concentrations of antidote oligonucleotides (see Fig. 11) were then added, and the APTT was measured at 10 minutes after antidote addition to plasma (Fig. 13A).
  • Each of the complimentary antidote oligonucleotides tested was able to reverse >90% of the anticoagulant activity of aptamer 9.3t-3NT within 10 minutes of addition in human plasma, with AS3NT-3 demonstrating the most potent reversal activity (Fig. 13B). This reversal activity is comparable to the ability of protamine to reverse the APTT increase following heparin addition to human plasma.
  • the reversal of the anticoagulant activity of 9.3t-3NT by antidote oligonucleotide AS3NT-3 was compared to the reversal of the anticoagulant activity of 9.3t by antidote oHgonucleotide Anti DI .
  • the addition of the tail to the aptamer increases the efficiency of reversal by an antidote oHgonucleotide with sequences complimentary to the 3' tail of the aptamer (Figs. 14A and 14B).
  • oligonucleotide modulators have been produced that target aptamer 9.3t, and are effective at reversing its anticoagulant activity in human plasma in vitro (all are 2'Omethyl oligonucleotides):
  • Anti D Tl 5' cau ggg gag gca gca uua 3' AS 9.3t-2: 5' cau ggg gag gca gca 3' AS 9.3t-3: 5' cau ggg gag gca 3'.
  • oligonucleotide modulators target aptamers 9.3t and 9.3t-3NT, and are effective at reversing its anticoagulant activity in human plasma in vitro (all are 2'Omethyl oligonucleotides):
  • AS 5-1 5' gca uua cgc ggu aua guc ecc ua 3'
  • AS 5-2 5' cgc ggu aua guc ecc ua 3'.
  • oligonucleotide modulators target either aptamer 9.3t or aptamer 9.3t-3NT, and are mutant oligonucleotides designed to test specific aspects of the design of modulating oligonucleotides to these aptamers (all are 2'Omethyl oligonucleotides):
  • AS 9.3t-M 5' cau ggg gaa gca 3' (SEQ LDNO:37)
  • AS 9.3t-3NOH 5' aug ggg agg ca 3' (SEQ ID NO:38).
  • AS 3NT-3M 5' gac aug ggg aag ca 3' (SEQ ID NO:39)
  • AS 3NT-3 MT 5' aca aug ggg agg ca 3' (SEQ ID NO:40)
  • the antidote oligonucleotide 5-2C (5' CGC GGU AUA GUC CCC AU) but not a scrambled version of this antidote oligonucleotide, 5-2C ser, has been shown to effectively reverse the activity of aptamers 9.3t and Peg-9.3t in vitro in human plasma.
  • Peg-9.3t is 9.3t with a 40 KDa polyethylene glycol appended to its 5 'end via a linker.
  • aptamer was added to plasma (50 nM 9.3t, 125 nM Peg-9.3t) and allowed to incubate for 5 minutes. Antidote oligonucleotides were then added, and APTT assays were initiated 10 minutes after antidote addition. The results are shown in Figure 15.
  • Aptamers 9.3t or Peg-9.3t were added to human plasma in vitro at a final concentration of 50 nM for 9.3t or 125 nM for Peg-9.3t, and allowed to incubate for 5 minutes at 37°C.
  • Antidote oligonucleotide 5-2C at the indicated molar excess to the aptamer was then added, and the residual aptamer activity was determined by measuring the clotting time in APTT assays at the times indicated following antidote addition.
  • the duration of the inactivation of the anticoagulant activity of Peg-9.3t by antidote oligonucleotide 5-2C was measured in vitro in human plasma. Briefly, Peg-9.3t was added to human plasma to a final concentration of 125 nM and allowed to incubate for S minutes. Antidote oligonucleotide 5-2C was then added at a 10 fold molar excess, or in a parallel experiment buffer alone was added in place of the antidote oligonucleotide, and the clotting time was measure in an APTT assays at various time points following antidote addition. The % residual anticoagulant activity was determined as above.
  • the APTT of untreated human plasma was also measured in parallel to establish a baseline clotting time at each time point. It was found that after 5 hours of incubation at 37°C, the APTT of the untreated plasma began to increase, indicating the loss of the clot forming activity of the plasma, and the experiment was thus stopped at 5 hours.
  • FIG. 18A Depicted in Figure 18A is an aptamer (designated 1 lF7t) against coagulation factor Xa that is a potent anticoagulant in vitro in human plasma.
  • FIG. 18B Depicted in Figure 18B is a mutant version of aptamer HF7t, designated HF7tM. Alterations of the identity of the positions shown in Fig. 18B leads to a > 1300 fold loss of affinity of the mutant aptamer for coagulation Fxa. Varying concentrations of aptamers 1 lF7t and 1 lF7tM were added to human plasma in vitro, and the clot time was then measured in a PT (Fig. 19A) or APTT assay (Fig. 19B).
  • Aptamer 1 lF7t is also a potent anticoagulant of human plasma when assays in PT clotting assays, as would be expected for a FXa inhibitor.
  • the mutant aptamer, 1 lF7tM showed no anti coagulation activity in either the PT or APTT assay.
  • antidote oligonucleotides were screened for the ability to reverse the anticoagulant activity of aptamer 1 lF7t in vitro in human plasma:
  • FIG. 20 shows the sequences of 1 lF7t to which these antidote oligonucleotides are complimentary. As shown in Figure 21 A, the antidote oligonucleotides effectively reverse the activity of aptamer 1 lF7t in human plasma. In these experiments, aptamer was added to plasma (final concentration 125 nM) and allowed to incubate for 5 minutes.
  • Antidote oligonucleotides were then added, and APTT assays were initiated 10 minutes after antidote addition.
  • APTT assays were initiated 10 minutes after antidote addition.
  • the following sequences were also found to have antidote activity against aptamer 1 lF7t:
  • AO 3-2 5' CAAGAGCGG GGC CAAG3' AO 5-3: 5' CGAGUAUUAUCUUG3'.
  • Figure 2 IB shows the characterization of antidote 5-2 activity over a larger concentration range of antidote 5-2, and comparison to the antidote activity of a scrambled sequence version of antidote 5-2, 5-2scr.
  • the data demonstrate potent reversal activity of antidote 5-2, and specificity of antidote oligonucleotide activity as demonstrated by lack of reversal activity of AO 5-2scr.
  • Figures 22 and 23 relate to the ability to rapidly and durably control the anticoagulant activity of the FXa antagonist aptamer 1 lF7t, and its derivatives, using antidote oligonucleotides. Together these data demonstrate that the onset of action of the antidote is rapid (Fig. 22), that the time needed for the antidote to act is at least in part dependent on the antidote concentration (Fig. 22), and that once the antidote has inactivated the aptamer, this effect is durable (Fig. 23).
  • antidote oligonucleotides can be intravenously infused into a human or other animal would be a potential method to use antidote oligonucleotides to modulate the activity of a therapeutic aptamer. Furthermore, because the antidote activity is durable, once the desired level of modulation of the aptamer by the antidote is achieved, infusion of the antidote can be terminated, allowing residual antidote to clear the human or animal. This allows for subsequent re-treatment of the human or animal with the aptamer as needed. EXAMPLE 8
  • nucleic acid ligand 1 e.g., 9.3t
  • results demonstrate the utility of nucleic acid Hgand-antidote pairs for target validation, and the study of biochemical pathways.
  • the antidote enables one to determine that the response observed after inhibiting a target protein with an nucleic acid ligand is due to specifically inhibiting that protein.
  • the antidote makes it possible to determine if binding of the nucleic acid ligand to the target protein leads to protein turnover. For example, if upon antidote addition complete protein activity is restored, it argues that there was no net change in protein concentration as a result of nucleic acid ligand binding.
  • HIT heparin-induced thrombocytopenia
  • This disorder is characterized by a decrease in the platelet count and an increased risk for new or recurrent life and limb-threatening thromboembolism (Warkentin et al, Thromb Haemost 19: 1-7. (1998))
  • Several alternative anticoagulants are available, but none of these anticoagulants can be controlled by a reversing agent. This significantly limits the treatment options for patients with HIT, and hemorrhagic complications and recurrent thromboembolism while undergoing treatment are common Greinacher et al, Circulation 99:13- 80. (1999), Lewis et al, Circulation 103:1838-1843. (2001)).
  • aptamer Peg-9.3t and antidote 5-2C to serve as an anticoagulant-antidote pair in plasma samples from six patients with HIT was investigated, three with end-stage renal disease requiring hemodialysis and thus repeated anticoagulation, and three with thromboembolic complications requiring anticoagulant therapy.
  • serologic criteria included a positive heparin-induced platelet aggregation assay (Ortel et al, Thromb Haemost 67:292-296. (1992)) and/or elevated heparin/platelet factor 4 antibody levels detected by ELISA (GTI, Inc., Brookfield, WI).
  • aptamer 1 lF7t and it's corresponding antidote 5-2 serve as an anticoagulant-antidote pair in plasma samples from 2 patients with HIT, one with end-stage renal disease requiring hemodialysis and thus repeated anticoagulation, and one with thromboembolic complications requiring anticoagulant therapy.
  • Aptamer 1 lF7t prolonged the APTT clotting times of plasma from both patients, and antidote 5-2 was able to effectively reverse this anticoagulant activity to the pre-treatment baseline of each patient (Fig. 26).

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Abstract

Selon l'invention, l'activité biologique du ligands d'acides nucléiques est régulée (c'est-à-dire, renforcée ou inhibée) in vivo afin de produire un effet biologique souhaité. A cet effet, on administre un modulateur ou un régulateur qui change la liaison du ligand d'acides nucléiques pour sa cible ou qui dégrade, clive, métabolise ou décompose le ligand d'acides nucléiques pendant que ligand continue d'exercer son effet. Les modulateurs selon l'invention peuvent être administrés en temps réel selon le cas, en fonction de différents facteurs, notamment l'évolution du patient ou l'appréciation du médecin pour ce qui est des moyens d'appliquer un traitement optimal. Par conséquent, l'invention fournit, pour la première fois, un régime thérapeutique régulable au cours d'un traitement au ligand d'acide nucléiques.
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EP1601681A2 (fr) * 2003-03-12 2005-12-07 Duke University Mimetiques d'oligomeres
WO2005106042A3 (fr) * 2004-04-22 2006-12-07 Regado Biosciences Inc Modulateurs ameliores des facteurs de coagulation
US7514543B2 (en) 2002-06-07 2009-04-07 Trigen Gmbh Polynucleotides encoding immunoadhesins comprising a glycoprotein VI domain
US7531178B2 (en) 2002-06-07 2009-05-12 Trigen Gmbh Immunoadhesin comprising a glycoprotein VI domain
WO2009061841A2 (fr) * 2007-11-05 2009-05-14 Isis Pharmaceuticals, Inc. Antidotes à des composés antisens
CN101453891A (zh) * 2006-05-26 2009-06-10 雷加多生物科学公司 Reg1抗凝系统的给药
WO2010000665A1 (fr) * 2008-06-30 2010-01-07 Santaris Pharma A/S Oligomères d’antidote
US7741307B2 (en) 2000-09-26 2010-06-22 Duke University RNA aptamers and methods for identifying the same
WO2010094900A1 (fr) 2009-02-19 2010-08-26 Lfb Biotechnologies Moyens pour la purification d'une protéine de la coagulation, et procèdes pour sa mise en oeuvre
WO2010094899A1 (fr) 2009-02-19 2010-08-26 Lfb Biotechnologies ACIDES NUCLEIQUES SE LIANT SPECIFIQUEMENT AU FACTEUR VII/VIIa HUMAIN, ET UTILISATIONS
WO2010141771A1 (fr) * 2009-06-03 2010-12-09 Regado Biosciences, Inc. Acides nucléiques modulateurs de la glycoprotéine vi
WO2011012830A1 (fr) 2009-07-31 2011-02-03 Lfb Biotechnologies Procede pour la purification de proteines de la coagulation a domaine gla
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US7300922B2 (en) 2007-11-27
JP4773684B2 (ja) 2011-09-14
JP5834028B2 (ja) 2015-12-16
PT2070939E (pt) 2014-06-09
US8586524B2 (en) 2013-11-19
US20030083294A1 (en) 2003-05-01
US20080051339A1 (en) 2008-02-28
EP2364990A1 (fr) 2011-09-14
EP2070939B1 (fr) 2014-04-02
HK1131623A1 (en) 2010-01-29
EP1401853A4 (fr) 2004-12-15
US8283330B2 (en) 2012-10-09
ES2474192T3 (es) 2014-07-08
DK1401853T3 (da) 2010-11-01
JP2013139450A (ja) 2013-07-18
DK2070939T3 (da) 2014-06-23
US20100249217A1 (en) 2010-09-30
IL159053A (en) 2010-12-30
SG157961A1 (en) 2010-01-29
CY1110978T1 (el) 2015-06-11
EP1401853B1 (fr) 2010-09-22
AU2009200036B2 (en) 2012-07-26
CA2448567A1 (fr) 2002-12-05
AU2002312059B2 (en) 2009-01-15
CA2448567C (fr) 2014-05-20
SG2011071982A (en) 2016-09-29
EP2070939A1 (fr) 2009-06-17
IL159053A0 (en) 2004-05-12
AU2009200036A1 (en) 2009-02-12
US20140275225A1 (en) 2014-09-18
JP2005502596A (ja) 2005-01-27
EP1401853A1 (fr) 2004-03-31
JP2009185035A (ja) 2009-08-20
DE60237768D1 (de) 2010-11-04
PT1401853E (pt) 2010-12-07
ATE482227T1 (de) 2010-10-15

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